Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [³²P]P_i into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [³²P]P_i into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [³²P]P_i into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.     

In vitro studies of skeletal muscle membranes characterization of a phosphorylated intermediate of sarcolemmal (Na+ + K+)ATPase

The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating ³²P from [γ?³²P]ATP. The membrane protein phosphorylation requires Mg²⁺. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation.The Mg²⁺-dependent ³²P labeling was significantly enhanced by Na⁺. The rate of Na⁺ -stimulated ³²P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K⁺ reduced the Na⁺ -stimulated ³²P-incorporation but enhanced the ³²P_i release. This inhibitory effect of K⁺ on Na⁺ -stimulated ³²P incorporation was prevented by the cardiac glycoside, ouabain.The Na⁺ -dependent ³²P labeling showed substrate dependency and the Na⁺ site was saturable. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP was 2 · 10?5 M. The optimum pH for ³²P labeling was between 7 and 8.Na⁺ -dependent membrane phosphorylation showed a direct relationship with the (Na⁺ + K⁺ATPase activity. The high turnover rate of ³²P intermediate (12 000 min ?1) suggested its functional significance in the overall transport ATPase reaction sequence.The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ³²P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of ³²P incorporation was also observed into a 25 000 dalton subunit and Ca²⁺ slightly enhanced the phosphorylation of this component.The size ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{108 000}$) and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na⁺ + K⁺ATPase preparations reported so far.     

Phosphatidylinositol turnover in mitogen-activated lymphocytes. Suppression by low-density lipoproteins

Low-density (LD) lipoproteins inhibit phytohaemagglutinin-enhanced turnover of phosphatidylinositol in human peripheral lymphocytes. Turnover was assessed by ³²P incorporation into phospholipids and by loss of ³²P from [³²P]phosphatidylinositol. Inhibition of lipid turnover by LD lipoproteins is not the result of a change in the amount of phytohaemagglutinin required for maximum cellular response. Neither phytohaemagglutinin nor LD lipoproteins influence ³²P incorporation into phosphatidylethanolamine and phosphatidylcholine during the first 60min after mitogenic challenge. The extent of inhibition of phosphatidylinositol turnover by LD lipoproteins depends on the concentration of LD lipoproteins present in the incubation medium: 50% of maximum inhibition occurs at a low-density-lipoprotein protein concentration of 33μg/ml and maximum inhibition occurs at low-density-lipoprotein protein concentrations above 100μg/ml. Phytohaemagglutinin stimulates ³²P incorporation into phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. However, LD lipoproteins abolish ³²P incorporation into phosphatidylinositol without affecting incorporation into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The ability of LD lipoproteins to inhibit phytohaemagglutinin-induced phosphatidylinositol turnover is mimicked by EGTA. Furthermore, inhibition of LD lipoproteins by phytohaemagglutinin-induced ³²P incorporation into phosphatidylinositol correlates directly with inhibition by LD lipoproteins of Ca²⁺ accumulation. These results suggest that Ca²⁺ accumulation and turnover of phosphatidylinositol are coupled responses in lymphocytes challenged by mitogens. The step in phosphatidylinositol metabolism that is sensitive to LD lipoproteins and, by inference, that is coupled to Ca²⁺ accumulation is release of [³²P]phosphoinositol from phosphatidylinositol.     

Effects of Ca2+, Mg2+, and depolarizing agents,on the32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes

In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the³²Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A₂ inhibits the labeling of PIP₂ and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K⁺ or 100 M veratrine decreases the labeling of PIP₂ and PIP by 66–74%. The decreased labeling results in large part from the Ca²⁺-dependent degradation of³²P-labeled PIP₂ and PIP as shown by pulse-chase experiments in which PIP₂ and PIP were prelabeled with³²Pi. Depolarization of synaptosomes results in the stimulation of⁴⁵Ca²⁺ uptake with the concomitant hydrolysis of PIP and PIP₂. Addition of 1 mM Ca²⁺ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K⁺ accounts for 75% of the enhanced degradation of PIP₂ and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of⁴⁵Ca²⁺ uptake. EGTA (10mM) and Mg²⁺ (5–10 mM) inhibit the degradation of PIP and PIP₂ and counteract the action of 1 mM Ca²⁺. Our data demonstrate that⁴⁵Ca²⁺, Mg²⁺, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP adenosine triphosphate - Pi inorganic orthophosphate - PIP phosphatidylinositol-4-phosphate - PIP₂ phosphatidylinositol-4,5,-bisphosphate - IP₃ inositol-1,4,5-trisphosphate     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

Cyclic AMP independent protein kinase activity in rat cerebral cortex synaptic vesicles—Partial characterization

The intrinsic protein kinase activity of a highly purified synaptic vesicle preparation was characterized. The time-course of the reaction was found to be rapid and linear for about 1 min, but plateaued after 30 min by which time approximately 1 nmol of³²P pering protein was incorporated into trichloroacetic acid precipitated vesicular protein. The enzyme was optimally active at pH 6.0 (37°C), and had apparentK _m values of 40 and 88 M for ATP and GTP respectively. The enzyme was not stimulated by cAMP or cGMP. Mg²⁺ was required for maximal activity. The reaction was inhibited by free Ca²⁺, and non-selectively by Na⁺, K⁺, and NH₄ ⁺.Deceased.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

Effects of dopamine, gamma-aminobutyric acid and 5-hydroxytryptamine on incorporation of 32P into phosphatides in slices from the guinea pig brain

(1) Dopamine–In slices from guinea pig corpus striatum, dopamine significantly inhibited incorporation of ³²P into phosphatidylethanolamine-plus-phosphatidylserine at a concentration of 0001 mM, and into phosphatidylinositol and phosphatidylcholine at 001 mM. In eight areas of the guinea pig brain in which the effects of 01 mM-dopamine were studied, the only significant increase in incorporation of ³²P into phosphatides was into phosphatidic acid in the hypothalamus; there was significant inhibition of incorporation of ³²P into phosphatidylcholine in cerebellar cortex and thalamus, and into phosphatidylethanolamine-plus-phosphatidylserine in the olfactory bulbs. (2) Gamma-aminobutyric acid—In slices of guinea pig cerebral cortex, GABA (1 mM) significantly inhibited incorporation of ³²P into only phosphatidic acid, diphosphoinositide and phosphatidylinositol and did not significantly affect the level or the specific activity of the nucleotide ～P. GABA (10 mM), significantly inhibited incorporation of ³²P into diphosphoinositide, phosphatidylinositol and phosphatidylcholine, and significantly lowered the specific activity of the nucleotide ～P. (3) 5-Hydroxytryptamine—In slices of guinea pig cerebral cortex, 5HT, (1 mM) significantly increased incorporation of ³²P into phosphatidic acid; in a concentration of 10 mM, 5HT increased incorporation of ³²P into phosphatidic acid four-fold and into both diphosphoinositide and phosphatidylinositol two-fold; other phosphatides were not significantly affected and the specific activity of the nucleotide ～P was not significantly different. In eight brain areas studied, 5HT (10 mM) significantly increased incorporation of ³²P into phosphatidic acid in all areas; into phosphatidylinositol in six areas (excepting cerebellar cortex and hypothalamus); and into diphosphoinositide in the olfactory bulbs, cerebral cortex, hypothalamus and corpus striatum. Incorporation of ³²P into triphosphoinositide was not significantly affected in any area. Incorporation of ³²P into phospha-tidylethanolamine-plus-phosphatidylserine was significantly greater than the control in the olfactory bulbs and incorporation of ³²P into phosphatidylcholine was significantly less than the control in the cerebellar cortex, olfactory bulbs and hypothalamus. (4) The possibility is discussed that increased incorporation of ³²P into phosphatidic acid and/or phosphatidylinositol in response to neurotransmitters might be associated with excitatory, but not inhibitory, neurotransmission; and that inhibition of incorporation of ³²P into various phosphatides may be associated with inhibitory neurotransmission or neuromodulation.     

Studies on the effects of acetylcholine and antiepileptic drugs on32Pi incorporation into phospholipids of rat brain synaptosomes

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated³²P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated³²P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the³²P labeling of phospholipids nor on the specific radioactivity of [³²P]ATP. Omission of Na⁺ drastically reduced both the³²P labeling of synaptosomal phospholipids and the specific radioactivity of [³²P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate Na⁺ and Ca²⁺ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca²⁺ and Na⁺ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.Abbreviations used ACh Acetylcholine - PA phosphatidic acid - PI phosphatidylinositol - poly PI polyphosphoinositides (diphosphoinositide and triphosphoinositide) - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - S.A. specific radioactivity     

Effect of Short-Term Salinity on Lipid Metabolism and Ion Accumulation in Tomato Roots

To examine the ion accumulation and membrane lipid metabolism in response to salinity we compared two tomato cvs. Pera and Hellfrucht Fruhstamm (HF), considered to be salt-tolerant and sensitive respectively. Na⁺ and K⁺ accumulation was significantly higher in roots of cv. Pera after 24 h and 72 h of 100 mM NaCl. While in cv. HF, a temporary increase in K⁺ accumulation at 24 h was accompanied by a sustained increase in Na⁺ content. Both cultivars enhanced incorporation of [³²P]orthophosphate into phosphatidylinositol 4,5-bisphosphate at 24 h and 72 h of NaCl. In parallel to the increase of phosphatidylinositol 4,5-bisphosphate a decrease in phosphorylation of phosphatidic acid and phosphatidylcholine were observed in the sensitive cv. HF. Structural and signal lipid changes in response to salinity were more evident in the sensitive cv. HF. Salt tolerant cv. Pera accumulated Na⁺ ions in the roots without considerable modifications in lipid metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Brain asymmetry in phospholipid polar head group metabolism: Parallel in vivo and in vitro studies

Phospholipid content and³²P-incorporation have been studied in individual rat cerebral hemispheres. The total phospholipid content was 44.9±0.9 and 47.9±1.3 mol lipid P/100 mg protein for the right and left hemispheres respectively. Individually, only sphingomyelin was significantly (about 30%) higher in the left hemisphere. Metabolic experiments have been conducted in vivo using i.p. injection of³²P and following its incorporation into total and individual phospholipids in each cerebral hemisphere. Higher incorporations were attained by phosphatidate and phosphatidylinositol-4,5-bisphosphate (PIP₂) in the left cerebral hemisphere than in the right. In an attempt to determine whether phospholipid metabolism is also lateralized in specific subcellular compartments related with the neurotransmission process, we have studied in vitro the [³²P] incorporation into phosphoglycerides of synaptosomal fractions obtained from each cerebral cortex. The precursor was taken up differently by the two cerebral cortex preparations, resulting in different profiles of distribution among lipids. In addition, the kinetics of lipid labeling showed higher rates of³²P-incorporation in fractions derived from the left cerebral cortex, mainly in PIP and PIP₂, These results are interpreted to indicate that several enzymes involved in lipid metabolism are modulated to a different extent in the two hemispheres.     

PHOSPHOLIPID METABOLISM IN CULTURED NEUROBLASTOMA AND GLIOMA CELLS INCUBATED WITH CARBAMYLCHOLINE AND NOREPINEPHRINE

Cultures of cloned neuroblastoma cells (N1E) in stationary phase and cloned glioma cells (C2₁) in confluency showed substantial differences in phospholipid composition. As a percentage of lipid P, N1E contained more phosphatidylcholine, less ethanolamine phosphoglycerides and much less sphingomyelin than C2₁. When incubated with ³²P_i both cell lines incorporated comparable amounts of radioactivity into total phospholipids. In NIE, phosphatidylcholine contained much more and phosphatidylinositol and phosphatidic acid somewhat less label as compared to C2₁. The presence in the incubation medium of either norepinephrine or carbamylcholine failed to elicit stimulation of ³²P incorporation into any phospholipid class.     

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K⁺ concentrations ([K⁺] _o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V _i) were the same for different [K⁺] _o . For the range ofV _i examined, K⁺ efflux occurs principally through the K⁺ inward rectifier channels. When external K⁺ is removedV _i remains constant for 2 to 3 hr because of the high membrane conductance to Cl^–, but K⁺ efflux drops by about one order of magnitude.External Ba²⁺ in the presence or absence of external K⁺ produces an inhibition of K⁺ efflux described by a relation of the formu=(u₁/(1+C)[Ba²⁺] _o ))+u ₂, whereu is the uninhibited fraction of K⁺ efflux;u ₁, u₂ andC are constants; andu ₁+u₂=1.C depends both on [K⁺] _o andV _i. When [K⁺] _o 75mm, increasing [K⁺] _o at constantV _i reduces Ba²⁺ sensitivity. For constantV _i–30 mV, Ba²⁺ sensitivity is less when [K⁺] _o =0 than when [K⁺] _o 75mm. When [K⁺] _o =0, Ba²⁺ sensitivity decreases asV _i is made more positive. The dependence of the Ba²⁺ sensitivity onV _i at constant [K⁺] _o is greater when [K⁺] _o =0 than when [K⁺] _o 75mm.Both the activation of K⁺ efflux by external K⁺ and the Ba²⁺ inhibition of K⁺ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K⁺, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba²⁺ occupies either site, K⁺ efflux is further reduced. The reduction of Ba²⁺-sensitivity by increasing [K⁺] _o at high [K⁺] _o is attributable to the displacement of Ba²⁺ from the control sites by K⁺. The increased Ba²⁺ sensitivity produced by going from [K⁺] _o =0 to [K⁺] _o >-75mm whenV _i–30 mV is attributable to states in which Ba²⁺ occupies one site and K⁺ the other when [K⁺] _o 0. The smallerV _i dependence of the Ba²⁺ sensitivity when [K⁺] _o 75mm compared to [K⁺] _o =0 is attributable to the necessity that Ba²⁺ displace K⁺ at the control sites when [K⁺] _o is high but not when [K⁺] _o =0.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

A physicochemical study of the interaction of phosphatidylinositol with buprenorphine and naloxone

The interactions of two opioid molecules (buprenorphine and naloxone) with phosphatidylinositol and phosphatidylcholine were studied in lipid monolayers at the air-water interface. The influence of Na⁺, Ca²⁺, and Mn²⁺ ions in these interactions has also been determined. Neither buprenorphine nor naloxone influence the ordered state of phosphatidylcholine monolayers. On the contrary, both opioid molecules interact specifically with phosphatidylinositol monolayers. The area/molecule of phosphatidylinositol spread on buprenorphine containing subphases is highly affected by this molecule and also by ions. The phosphatidylinositol/naloxone interactions are rather weak and less affected by ions.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - Bup Buprenorphine - Nx naloxone - ODS octadecyl silica - k capacity factor - logP partition coefficient between octanol and water     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Summary Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the³²Pi-incorporation into phosphoinositides. The extent of³²Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP₂) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate³²Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP₂, PIP and PI and increased PA labeling. Li⁺ also inhibited the microwave stimulated PIP₂, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A₂₃₁₈₇, inhibited the basal and microwave stimulated³²Pi labeling of PIP and PIP₂, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP₂ labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.