Calciotropic hormones raise the chemically detectable [Pi] in UMR 106–06 osteoblast-like cells

Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10^?11 to 10^?7 M ) increases both ³²Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D₃ (1,25D) (10^?12 to 10^?6 M ) and salmon calcitonin (CT) (10^?12 to 10^?6 g ml^?1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular ³²Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D₃. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth.     

Skeletal muscle Pi transport and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles.

In the rat skeletal myoblast line L6 and in a rabbit skeletal muscle sarcolemma/t-tubule vesicle preparation, [32P]Pi uptake was largely dependent on the transmembrane Na gradient. Na-dependent [32P]Pi uptake had a hyperbolic relationship to [Pi] and [Na], being half-maximal at 0.2-0.3 mM [Pi] and at 25-40 mM [Na]. In vesicles the Na-dependence suggests that approx. two Na are transported with each Pi, but the inhibition of [32P]Pi uptake at high pH suggests that the Pi monoanion is the transported form. Together these imply electrogenic transport and this is confirmed by the results of manipulating the vesicle membrane potential. Thus, electrogenic Na-Pi co-transport exploits both the sodium gradient and the cell membrane potential to maintain muscle cellular [Pi] against an unfavourable electrochemical gradient. The low [Pi] for half-maximal flux may partly explain the small effect of altered extracellular [Pi] on cellular [Pi]. In L6 myoblasts most 32P was first detectable in an organic phosphate pool rather than cellular Pi, while the specific activity of cell Pi rapidly reached 40% of that of extracellular Pi and was stable for at least 3 h. These results are discussed in terms of the organisation of cellular phosphate metabolism.     

Phosphate uptake in Chara: membrane transport via Na/Pi cotransport

Phosphate uptake in the freshwater charophyte plant Chara corallina was found to be strongly dependent on the presence of Na in the external medium. Based on the reciprocal stimulations of ³²Pi uptake by Na and ²²Na uptake by Pi, the logical mechanism for Pi uptake appears to be a nNa/Pi symport with a half‐maximal stimulation (K_m) for Na of approximately 300 μM and a K_m for Pi of approximately 10 μM . Comparison of the stimulations of ³²Pi and ²²Na influxes at pH 6 gives a stoichiometry of Na : Pi of 5·68. The reduction in Pi influx with increasing pH is consistent with the transported species being the monovalent H₂PO₄^?. In voltage‐clamp experiments, currents elicited by Pi in the presence of Na were equivalent to an influx of positive charge which exceeded the measured influxes of ³²P by a factor of 6·26. Intracellular perfusion was used to examine the dependence of Pi influx on ATP and Na. In perfused cells, Pi influx was low when ATP was absent from the internal medium or Na was absent from the external medium. Addition of ATP alone had little effect whereas addition of Na alone increased the ³²Pi influx slightly. Addition of both ATP and Na together restored Pi influx to rates comparable to those of intact cells. It is suggested that the ATP is required for membrane hyperpolarization which in turn drives the highly electrogenic flux of Pi with up to 6 Na. However, consideration of the electrochemical potential differences for Na and Pi at pH less than 6 shows that nNa/Pi would not be feasible. It is suggested that at low pH, H⁺ may substitute for Na.     

Spectroscopic Characterization of a VO<Superscript>2+</Superscript> Complex of Oxodiacetic Acid and its Bioactivity on Osteoblast-like Cells in Culture

The oxovanadium(IV) complex of oxodiacetic acid (H₂oda) of stoichiometry [VO(oda)(H₂O)₂], which presents an unprecedented tridentate OOO coordination, was thoroughly characterized by infrared, Raman, electronic, and electron paramagnetic resonance spectroscopies. The biological activity of the complex on the cell proliferation and differentiation was tested on osteoblast-like cells (MC3T3E1 osteoblastic mouse calvaria-derived cells and UMR106 rat osteosarcoma-derived cells) in culture. The complex caused inhibition of cellular proliferation in both osteoblast-like cells in culture, but the cytotoxicity was stronger in the normal (MC3T3E1) than in the tumoral (UMR106) osteoblasts. The effect of the complex in cell differentiation was tested through the specific activity of alkaline phosphatase of the UMR106 cells because they expressed a high activity of this enzyme. What occurs with other vanadium compounds [VO(oda)(H₂O)₂] is an inhibitory agent of osteoblast differentiation.     

Die pH-Abhängigkeit der Aufnahme von H2PO 4 - , SO 4 = , Na+ und K+ und ihre gegenseitige Beeinflussung bei Ankistrodesmus braunii

Summary Ion uptake was studied using ³²P, ³⁵S, ²²Na and ⁴²K as tracers in synchronized cells of Ankistrodesmus, which were slightly starved with respect to the ions to be investigated. In the light and in the dark, phosphate uptake is maximal between pH 5.5 and 6.5. Whereas Na⁺ in comparison to K⁺ enhances phosphate uptake in the light (8 to 9-fold) and in the dark, Ca⁺⁺ exerts only a slightly stimulatory effect. The stimulation of phosphate binding by Na⁺ occurs rapidly, even after less than 5 sec of incubation, and also in the presence of an equimolar concentration of K⁺.The pH-dependence of Na⁺-uptake in the light and in the dark is comparable to a dissociation curve: Na⁺-uptake increases with decreasing extracellular H⁺-concentration and is inversely proportional to phosphate uptake in the absence of Na⁺. The light:dark ratio of Na⁺-uptake at pH 8 amounts to 7:1. Mere adsorption of Na⁺ is similarly dependent on the pH. K⁺ strongly competes with Na⁺-uptake, even at pH 8. K⁺-uptake proceeds in a quite different manner from Na⁺-uptake and has an optimum at pH 7.Sulfate is taken up linearly in a biphasic process as a function of time; the pH-optimum lies between pH 7.5 and 8. K⁺ but not Na⁺ slightly enhances sulfate uptake.The Na⁺-enhancement of phosphate uptake can be related neither to a sodium-potassium exchange pump nor to a photosynthesis-dependent ion-exchange reaction.The results suggest that the uptake of phosphate, Na⁺ and K⁺, and the influence of alkali cations on phosphate uptake, but not sulfate uptake, are strongly dependent on fixed charges of the plasmalemma or even of the cell wall. These fixed charges may even prevent an active ion uptake.     

Induction of the Na+/Pi cotransport system in the plasma membrane of Chara corallina requires external Na+ and low levels of Pi

It was shown in previous studies that the giant freshwater alga Chara corallina does not control its Na⁺‐dependent Pi uptake by monitoring the internal Pi concentration and it was hypothesized that Chara may instead detect changes in Pi supply from the environment. The present work investigated the conditions that control the induction and inactivation of high affinity Na⁺/Pi influx in Chara. Withdrawal of Pi from the external medium resulted in a gradual increase in the rate of uptake measured immediately after Pi was resupplied. The increase continued for at least 7 d of starvation. In the initial stages, 0·5 or 1 µm Pi were more effective at inducing transport activity than no Pi, suggesting that low levels of Pi are actually required for induction. The high Na⁺‐dependent Pi uptake observed in Pi‐starved cells was inactivated by treatment with as little as 1 µm Pi over 6 d. External Na⁺ plays a major role in controlling the capacity for Na⁺/Pi cotransport activity, and in the absence of Na⁺, both induction and inactivation were either delayed or abolished. Na⁺ starvation stimulated Na⁺ uptake even though there were no measurable changes in the concentrations of Na⁺, or of K⁺ or Pi in either the vacuole or cytoplasm. It was concluded that both substrate (Pi) and driver ion (Na⁺) are required at adequate concentrations for the induction of the cotransporter. In the case of Pi, it was suggested that passive leakage of Pi from the cell into the apoplast is sufficient for this purpose but that supplementation by up to 1 µm Pi is more effective at the earlier stage. A mechanism for sensing the external supply of Pi is proposed.     

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.     

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀ = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V _max of the reaction (K _i = 7.6 ± 3 μM), having negligible effect on K _M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.     

Interactions of [14C]phosphonoformic acid with renal cortical brush-border membranes. Relationship to the Na+-phosphate co-transporter

Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [14C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [14C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by -80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [14C]PFA binding, but it did not change the maximum binding capacity. The maximum [14C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [14C]PFA binding correlated (r = 0.98; p less than 0.01) with percent inhibition by an equimolar dose of PFA of the (Na+o greater than Na+i)-dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [14C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [14C]PFA binding on BBMV. Bound [14C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally (r = 0.99; p less than 0.05) to their ability to inhibit (Na+o greater than Na+i)-gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [14C]PFA binding. The Na+-dependent binding of [14C]PFA and of [3H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [14C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter.     

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

The effects of parathyroid hormone (PTH) on cytoplasmic free CA²⁺ (Ca _i ²⁺ ) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01.In fura-2 loaded adherent single cells bPTH 1-34 (10 nM–1M) induced a rapid transient increase in Ca _i ²⁺ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 M) induced a transient increase in Ca _i ²⁺ in 20% of the experiments. The transient increase in Ca _i ²⁺ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Ca _i ²⁺ was derived from intracellular stores.A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Ca _i ²⁺ .These data show that PTH causes a release of Ca²⁺ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Ca _i ²⁺ in osteoblasts.     

Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants

Phosphate in the xylem exudate of tomato (Lycopersicon esculentum) plants was 70 to 98% inorganic phosphate (Pi), 2 to 30% P-choline, and less than 1% P-ethanolamine. Upon adding ³²Pi to the nutrient, Pi in xylem exudate had the same specific activity within 4 hours. P-choline and P-ethanolamine reached the same specific activity only after 96 hours. The amount of Pi in xylem exudate was dependent on Pi concentration in the nutrient and decreased from 1700 to 170 micromolar when Pi in the nutrient decreased from 50 to 2 micromolar. The flux of 0.4 nmoles organic phosphate per minute per gram fresh weight root into the xylem exudate was not affected by the Pi concentration in the nutrient solution unless it was below 1 micromolar. During 7 days of Pi starvation, Pi in the xylem exudate decreased from 1400 to 130 micromolar while concentrations of the two phosphate esters remained unchanged.

The concentration of phosphate esters in the xylem exudate was increased by addition of choline or ethanolamine to the nutrient solution, but Pi remained unchanged. Upon adding [¹⁴C]choline to the nutrient, 10 times more [¹⁴C]P-choline than [¹⁴C]choline was in the xylem exudate and 85 to 90% of the ester phosphate was P-choline. When [¹⁴C]ethanolamine was added, [¹⁴C]P-ethanolamine and [¹⁴C]ethanolamine in the xylem sap were equal in amount. P-choline and P-ethanolamine accumulated in leaves of whole plants at the same time and the same proportion as observed for their flux into the xylem exudate. No relationship between the transport of P-choline and Pi in the xylem was established. Rather, the amount of choline in xylem exudate and its incorporation into phosphatidylcholine in the leaf suggest that the root is a site of synthesis of P-choline and P-ethanolamine for phospholipid synthesis in tomato leaves.

     

Effects of medium amino acids on ouabain-sensitive 86Rb+ -uptake and membrane-potential dependent [3H]tetraphenylphosphonium accumulation in Friend erythroleukemia cells

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.     

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0- 1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene- 2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.     

Inorganic phosphate modulates responsiveness to 24,25(OH)2D3 in chondrogenic ATDC5 cells

Chondrogenic ATDC5 cells were used as a model of in vitro endochondral maturation to study the role of inorganic phosphate (Pi) in the regulation of growth plate chondrocytes by vitamin D3 metabolites. ATDC5 cells that were cultured for 10 days post‐confluence in differentiation media and then treated for 24 h with Pi produced a type II collagen matrix based on immunohistochemistry and expressed mRNAs for several chondrocytic markers, including aggrecan, collagen types II and X, cartilage oligomeric matrix protein, and SOX9. Pi also caused a decrease in [³⁵S]‐sulfate incorporation and stimulated apoptosis, as evidenced by increased DNA fragmentation and caspase‐3 activity. In addition, treatment with Pi induced sensitivity to 24,25‐dihydroxyvitamin D3 and this effect was both dose‐dependent and was blocked by phosphonoformic acid (PFA), a specific inhibitor of sodium dependent type III Pi transporters. Treatment with 24R,25(OH)₂D₃ reduced cell number and increased alkaline phosphatase specific activity in a dose‐dependent manner. Moreover, 24R,25(OH)₂D₃ reversed the Pi‐induced decrease in incorporation of [³H]‐thymidine and [³⁵S]‐sulfate incorporation, as well as the Pi‐induced increase in apoptosis. These results suggest that Pi acts as an early chondrogenic differentiation factor, inducing response to 24R,25(OH)₂D₃; treatment of committed chondrocytes with Pi induces apoptosis, but 24R,25(OH)₂D₃ mitigates these effects, indicating a possible inhibitory feedback loop. J. Cell. Biochem. 107: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Ion Fluxes and Phytochrome Protons in Mung Bean Hypocotyl Segments: II. Fluxes of Chloride, Protons, and Orthophosphate in Apical and Subhook Segments

The active form of phytochrome (Pfr) decreased CI⁻ uptake by subhypocotyl hook segments of Phaseolus aureus Roxb. and increased uptake by apical segments. Pfr had similar effects on Pi [³²Pi] uptake. Modulations of Pi [³²Pi] uptake were detectable 10 minutes following photoconversion. Pfr may modulate Pi influx across the plasmalemma. Pfr inhibited H⁺ extrusion by subhook segments and enhanced extrusion by apical hook segments. No rapid effects on H⁺ extrusion were found. Phytochrome may regulate a K⁺ -H⁺ exchange process. The differential responses of the two regions of the hypocotyl are discussed with respect to Pfr-mediated changes in growth and development.     

Effects of neurotransmitters and other pharmacological agents on 32Pi incorporation into phospholipids of the iris muscle of the rabbit

The effects of norepinephrine, other catecholamines, α- and β- adrenergic receptor blocking agents and acetylcholine on the incorporation of ³²Pi into phospholipids of the iris muscle of the rabbit were studied $\text{in vitro}$. There was a marked stimulation of ³²Pi into phosphatidic acid (PhA), phosphatidyl inositol (PhI) and to a much lesser extent phosphatidyl choline but not into phosphatidyl ethanolamine. The increase in the ³²P labeling of PhA and PhI in the presence of norepinephrine or acetylcholine, which ranged from 2- to 6-fold, was found to be time- and concerntration-dependent. Under our experimental conditions, several adrenergic drugs, including DL-propranolol, phentolamine, isoproterenol, phenylephrine, but not sotalol, increased markedly (nearly up to 5-fold) the ³²Pi incorporation into PhA and PhI of the iris. In contrast, phenoxybenzamine, an α-receptor blocker, blocked completely the stimulatory effects of norepinephrine on phospholipid synthesis. The stimulation of phospholipid synthesis by acetylcholine was completely abolished by atropine. Incorporation of ³²Pi into PhA and PhI was significantly increased in the presence of serotonin, dopamine, epinephrine or histamine. Addition of γ-aminobutyric acid or cyclic AMP was ineffective. These observations suggest that in the iris muscle of the rabbit, which is innervated by cholinergic and adrenergic fibers, the phospholipid effect is probably a membrane effect that is not associated with synaptic transmission.     

Inhibition of the veratridine-induced increase in cytosolic Ca2+ and respiration by Ca2+ antagonists in isolated cardiac myocytes.

1. We studied the effect of verapamil, nitrendipine, 3',4'-dichlorobenzamil (DCB) and Cd2+ on the increase in cytosolic free Ca2+ ([Ca2+]c) and the rate of O2-uptake induced by depolarization of isolated rat cardiac myocytes with veratridine. 2. The degree of inhibition by the several drugs tested on the increase in [Ca2+]c and respiration was dependent on extracellular Ca2+, pH and Na+. 3. Low verapamil and nitrendipine concentrations (2.5 microM) were fully effective in Ca2+ channel blockade, as indicated from experiments with isoproterenol and in a low-Na+ medium. 4. A complete inhibition of veratridine-induced increase in [Ca2+]c and O2-uptake was attained with higher Ca2+ blocker concentrations (25-30 microM), implying that these processes depend to a major extent on some other Ca2+ transport system, probably Na+/Ca2+ exchange.