Interaction of local anesthetics with small phospholipid vesicles investigated by proton NMR spectroscopy

The effects of the local anesthetics tetracaine, procaine (both charged at pH 6), and benzocaine (uncharged) on phospholipid liposomes have been investigated by 500 MHz ¹H NMR Spectroscopy. All the drugs reverse the Pr³⁺ induced shifts of phospholipid resonances in the same sequence as they are shifted by addition of Pr³⁺: choline POCH₂- > choline-CH₂N > choline-N(CH₃)₃ > glycerol > glycerol > acyl C₂ > acyl C₃. The drug effects result from incorporation of positive charges (tetracaine and procaine) and from the induction of a conformational change of the phospholipid head group via an action on the lipid glycerol backbone (benzocaine). From titration experiments with tetracaine on liposomes containing Pr³⁺ inside and outside is derived that the drug passes the bilayer by transverse diffusion. Tetracaine partitions outside/inside at a ratio of 21. Changes in linewidths of the drug resonances when incorporated into the liposomes allow the conclusion that the tetracaine molecule is located in an elongated way between the lipid acyl chains with its nitrogen group near the glycerol backbone. Benzocaine, showing strong effects on the line shapes of the protons on C₂ and C₃ of the lipid acyl chains is also located near the glycerol backbone, the region with the strongest hydrophobic forces.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 30), Cardiology.     

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     

Action of aspartate on the32Pi incorporation into phospholipids of cerebral cortex

The effect ofl-aspartate on the³²Pi incorporation of phospholipids, was studied on slices of rat cerebral cortex. This amino acid produced an inhibitory effect in concentrations 0.01–10 mM, which was more evident at 120 min. This effect was not stereospecific and did not imply a change in Pi uptake and in nucleotides P precursors. The inhibition was present in PS, PC, PE and to a lesser extent in PI. On liver slices 1 mMl-aspartate had the opposite effect, stimulating the incorporation of³²Pi into total phospholipids. Our results suggest that the effect ofl-aspartate is by a non-specific mechanism, probably not mediated by a receptor.     

Regulation of carrier-mediated and exocytotic release of [3H]GABA in rat brain synaptosomes

In this study we investigated the role of external monovalent cations, and of intracellular Ca²⁺ concentration ([Ca²⁺]i) in polarized and depolarized rat cerebral cortex synaptosomes on the release of [³H]--aminobutyric acid (³H-GABA). We found that potassium-depolarization, in the absence of Ca²⁺, of synaptosomes loaded with³H-GABA releases 7.4±2.1% of the accumulated neurotransmitter, provided that the external medium contains Na⁺, and an additional 19.0±2.5% is released upon adding 1.0 mM CaCl₂ to the exterior. The Ca²⁺-independent release component does not occur in a choline medium and it is only 3.4±0.8% of the³H-GABA accumulated in a Li⁺ medium, but both ions support the Ca²⁺-dependent release of³H-GABA (13.4±0.6% in choline and 15.4±1.5% in Li⁺), which suggests that the exocytotic release is independent of the external monovalent cation present, whereas the carrier-mediated release specifically requires Na⁺ outside. Furthermore, previous release of the cytosolic³H-GABA due to predepolarization in the absence of Ca²⁺ does not influence the amount of³H-GABA subsequently released by exocytosis due to Ca²⁺ addition (19.1±2.5% or 19.1±1.1%, respectively). In choline or Li⁺ medium, the value of the [Ca²⁺]i is raised by Na⁺/Ca²⁺ exchange to 663±75 nM or 782±54 nM, respectively, within three minutes after adding 1.0 mM Ca²⁺, in the absence of depolarization, and parallel release experiments show no release of³H-GABA in the choline medium, but a substantial release (7.1±2.1%) of³H-GABA occurs in the Li⁺ medium without depolarization. Subsequent K⁺-depolarization shows normal Ca²⁺-dependent release of³H-GABA in the choline medium (14.1±2.0%) but only 8.6±1.1% release in the Li⁺ medium, which suggests that raising the [Ca²⁺]i by Na⁺/Ca²⁺ exchange, without depolarization, supports some exocytotic release in Li⁺, but not in choline media. The role of [Ca²⁺]i and of membrane depolarization in the release process is discussed on the basis of the results obtained and other relevant observations which suggest that both Ca²⁺ and depolarization are essential for optimal exocytotic release of GABA.Special issue dedicated to Dr. Santiago Grisolia.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Features of cerebral support of verbal processes in children with dysgraphia and dyslexia

Comprehensive neuropsychological and electroencephalographic examination of children with dysgraphia and dyslexia demonstrated local EEG anomalies in various zones of the cortex in both hemispheres, mainly in the posterotemporal and/or anterior regions of the left and the posterotemporal regions of the right hemisphere. The character of speech disturbances depended considerably on the localization of the baseline EEG anomalies: disturbances of motor components of writing prevailed when these anomalies were localized in the anterior parts of the left hemisphere, while phonological and morphological speech disturbances were noted in children with local anomalies in the temporal regions of both hemispheres and in posterior association regions, mainly in the right hemisphere. Spectral and correlation analyses of the EEG demonstrated the features of the cerebral organization of verbal activity in children with dysgraphia and dyslexia, namely, weakened intra- and interhemispheric integration of electrical activity of different regions of the brain cortex, particularly temporal and posterior association regions, and predominance of integration based on low-frequency EEG components ( and ₁), unlike children without learning disabilities, in whom functional integration of cortical zones involved high-frequency rhythms (₂ and ). On the whole, the results of the study testify to an important role of systemic interactions between the regions of the cortex of both hemispheres in the formation of verbal activity.Translated from Fiziologiya Cheloveka, Vol. 31, No. 2, 2005, pp. 5–12.Original Russian Text Copyright © 2005 by Dmitrova, Dubrovinskaya, Lukashevich, Machinskaya, Shklovskii.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Characteristics of the norepinephrine-stimulated phosphatidylinositol turnover in rat pineal cell dispersions

Dispersed rat pineal cells can be used for the study of the phosphatidylinositol effect. The response to (–)-norepinephrine of the incorporation of³²P_i into phospholipids is linear with time and cell concentration, stereospecific, and mediated through ₁-adrenergic receptors. Na⁺ in the incubation medium is obligatory for labeling of phosphatidylinositol and phosphatidylcholine by³²P. In the absence of K⁺ incorporation of³²P is drastically lowered and no stimulation by norepinephrine occurs. Rb⁺ can replace K⁺. Omission of Ca²⁺ or substitution with Sr²⁺ preferentially lowers incorporation of radioactivity into phosphatidylcholine. Mg²⁺ is not required for basal or stimulated labeling.     

Imipramine-Induced Changes in 5-HT2 Receptor Sites and Inositoltrisphosphate Levels in Rat Brain

The effect of chronic administration of Imipramine on [³H]Spiperone binding to 5-HT₂ sites and inositoltrisphosphate (IP₃) levels in rat cerebral cortex was studied. Our data shows that treatment with imipramine (5 mg/kg body weight, intraperitoneally) for 30 days significantly down regulates 5-HT₂ receptors sites (262 ± 29 fmol/mg protein) in cerebral cortex (38%), compared to control rats (425 ± 60 fmol/mg protein., P < 0.001). However there was no significant change in the affinity of [³H]-Spiperone binding (kd) to 5-HT₂ sites in cerebral cortex after exposure to imipramine (Kd = 0.84 ± 0.11 nM). It is also observed that imipramine treatment significantly reduces 5-HT stimulated [³H]IP₃ formation in cerebral cortex (6,411 ± 708 dpm/mg protein), compared to the saline treated rats (12,238 ± 1,544 dpm/mg protein; P < 0.001), with concomitant decrease in Pdtlns-4–5-P₂. This study suggests that the therapeutic action of imipramine in brain might be by reducing hypersensitivity of 5-HT₂ receptors by down regulation, which leads to reduced levels of inositolphospholipids. This inturn reduces the levels of IP₃. In conclusion, imipramine acts at presynaptic site by blocking the reuptake of serotonin and at post synaptic site it downregulates 5-HT₂ sites with decreased IP₃ levels after chronic exposure.     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.     

The polyphosphoinositides,phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate,are present in nuclei isolated from carrot protoplast

Summary Nuclei were isolated from carrot protoplasts and the distribution of [³H]inositol-labeled phospholipids was analyzed by thinlayer chromatography. Phosphatidylinositol (PI), lysophos-phatidylinositol (LPI), phosphatidylinositol monophosphate (PIP), lysophosphatidylinositol monophosphate (LPIP), and phosphatidylinositol bisphosphate (PIP₂) were 55.7%, 12.3%, 5.0%, 11.5%, and 3.6% of the respective [³H]inositol-labeled lipids recovered from the nuclear fraction. While both the plasma membrane and nuclear fraction contained polyphosphoinositides, the distribution of the phosphoinositides and the amount of inositol-labeled lipid were distinct. For example, the nuclear fraction had a higher percentage of LPI and PIP₂ and less PI and LPIP than the plasma membrane fraction. The amount of [³H]inositol-labeled lipid recovered from the nuclear fraction per mg protein was an order of magnitude lower than that recovered from either the plasma membrane of lower phase fraction isolated by aqueous two-phase partitioning, or from whole cells and protoplasts. In addition, when the ratio of the [³H]inositol-labeled lipid was compared to total [¹⁴C]myristate-labeled lipid recovered there was three to ten fold less [³H] relative to [¹⁴C] in the nuclear fraction.These data indicate that while the polyphosphoinositides are a relatively high percentage of the inositol lipid in the nuclear fraction, the inositol lipid was only a small portion of the total lipid in the nuclei. Despite this low concentration of inositol lipid, when [ ³²P]-ATP was added to the isolated nuclei,³²P-labeled PIP and PIP₂ were synthesized. Thus, the carrot nuclei contained PI and PIP kinase as well as the polyphosphoinositides.Abbreviations PI phosphatidylinositol - LPI lysophosphatidylinositol - PIP phosphatidylinositol monophosphate - LPIP lysophosphatidylinositol monophosphate - PIP₂ phosphatidylinositol bisphosphate - DAG diacylglycerol - IP₃ inositol 1,4,5-trisphosphate     

Fertilization Stimulates an Increase in Inositol Trisphosphate and Inositol Lipid Levels inXenopusEggs

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for sperm-induced egg activation inXenopus laevis.Here we measure the endogenous production of both Ins(1,4,5)P₃and PIP₂during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P₃increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 μM) to a peak of 0.42 pmole per egg (0.93 μM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P₃during the time that the Ca²⁺wave is propagating across the egg suggests the involvement of Ins(1,4,5)P₃in wave propagation. This increase in Ins(1,4,5)P₃is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P₃involves a PIP₂hydrolysis pathway that is not simply raising intracellular Ca²⁺. While one might expect PIP₂levels to fall as a result of hydrolysis, we find that PIP₂actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP₂per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP₂concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP₂in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP₂as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP₂normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP₂concentrations, suggesting that the 2-fold higher total PIP₂in the vegetal half is not due to a gradient of PIP₂in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP₂.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

A simple,single-tube radioisotopic assay for the phosphorylation/inactivation activity of the pyruvate,orthophosphate dikinase regulatory protein

A simple, single-tube radiolsotopic method has been developed to assay the relative phosphorylation (inaetivation) activity of the bifunctional regulatory protein (RP) of C₄-leaf pyruvate,orthophosphate dikinase (PPDK) in desalted leaf homogenates and partially purified preparations. RP catalyzes the inactivation of maize PPDK by phosphorylation of Thr-456, utilizing [-P]ADP as the specific phosphoryl donor. Existing spectrophotometric and radioisotopic assays for the detection of RP activity are either relatively insensitive or labor-intensive and timeconsuming. We describe a modified radioisotopic assay that couples the synthesis of [-³²P]ADP by exogenous adenylate kinase with the subsequent RP-catalyzed [-³²P]ADP-dependent phosphorylation of exogenous maize PPDK. The incorporation of [-³²P] is dependent on the initial concentrations of ATP and PPDK, as well as the presence of active RP. Desalted leaf homogenates of C₃ species fail to catalyze ³²P incorporation into exogenous maize PPDK. Conversely, heterologous systems containing the maize target enzyme and leaf homogenats of other C₄ species result in PPDK-specific ³²P-incorporation. This simple radioisotopic assay is at least 40-times more sensitive than the routine spectrophotometric assay, and qualitatively exhibits comparable sensitivity and requires significantly less time than the currently available radioisotopic RP assay. The present assay reliably generates [-³²P]ADP and as such may be useful for studies of other systems requiring -labeled ADP, which is not commercially available.Abbrevlations Ap₅A P¹, P⁵-di(adenosine-5)-pentaphosphate - Bicine N,N-bis[2-hydroxyethyl]glycine - DTT dithiothreitol - PEI poly(ethyleneimine) - PEP phosphoenolpyruvate - PEPC PEP carboxylase (E.C.4.1.1.31) - PPDK pyruvate,orthophosphate dikinase (E.C.2.7.9.1) - RP PPDK regulatory protein     

Sodium channel activation does not alter lipid metabolism in cultured neuroblastoma cells

The interaction of voltage-sensitive Na⁺-channels and membrane lipid metabolism was examined by incubating cultured neuroblastoma cells with neurotoxins which alter the voltage-dependent relationship between the closed and open conformation of the channel protein. Guanidinium flux rate, a measure of Na⁺-channel activation, was increased 10-fold by the combined action of veratridine (100 M) and scorpion venom (28 g/ml). This response was completely blocked by tetrodotoxin (1 M). Under the same experimental conditions, the toxins did not increase the efflux of [³H]arachidonic acid from prelabeled cell membrane lipids or stimulate uptake of exogenous [³H]arachidonic acid. In addition, altering membrane fatty acid composition by incubating cells for 24 hr in a medium containing 50 M arachidonic or oleic acid did not alter guanidinium flux rates relative to that of control cultures. When cells were pulsed with³²Pi for 60 min and stimulated by veratridine plus scorpion venom for an additional 30 min, uptake of³²Pi into phosphatidylinositol as reduced; stimulating cells with bradykinin, a receptor agonist which activates the inositol cycle, promoted a 3.8 fold increase. Polyphosphoinositide turnover was not affected by Na⁺-channel activation, but was stimulated by bradykinin. These results suggest that voltage-sensitive Na⁺-channel activation in cultured neuroblastoma cells can function independent of membrane phospholipid and fatty acid metabolism.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

Inhibition by gamma-hexachlorocyclohexane of acetylcholine-stimulated phosphatidylinositol synthesis in cerebral cortex slices and of phosphatidic acid-inositol transferase in cerebral cortex particulate fractions

• 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.

     

The mechanism of nitrate transport across the tonoplast of barley root cells

Nitrate-selective microelectrodes were used to measure not only nitrate activity in the cytoplasm and vacuole of barley (Hordeum vulgare L.) root cells, but also the tonoplast electrical membrane potential. For epidermal cells, the mean cytoplasmic and vacuolar pNO₃ (-log₁₀ [NO₃]) values were 2.3±0.04 (n=19) and 1.41±0.03 (n=35), respectively, while for cortical cells, the mean cytoplasmic and vacuolar nitrate values were 2.58±0.18 (n=4) and 1.17±0.06 (n=13), respectively. These results indicate that the accumulation of nitrate in the vacuole must be an active process. Proton-selective microelectrodes were used to measure the proton gradient across the tonoplast to assess the possibility that nitrate transport into the vacuole is mediated by an H⁺/NO ₃ ^– antiport mechanism. For epidermal cells, the mean cytoplasmic and vacuolar pH values were 7.12±0.06 (n=10) and 4.93±0.11 (n=22), respectively, while for cortical cells, the mean cytoplasmic and vacuolar pH values were 7.24±0.07 (n=3) and 5.09±0.17 (n=7), respectively. Calculations of the energetics for this mechanism indicate that the observed gradient of nitrate across the tonoplast of both epidermal and cortical cells could be achieved by an H⁺/NO ₃ ^– antiport with a 11 stoichiometry.Abbreviations and Symbols G/F free-energy change for H⁺/NO ₃ ^– antiport - F Faraday constant - pH_c cytoplasmic pH - pH_v vacuolar pH - p[NO₃]_c log₁₀ (cytoplasmic [NO ₃ ^– ]) - P[NO₃]_v -log₁₀ (vacuolar [NO₃]) We wish to thank Dr. K. Moore for assistance with statistical analysis.     

Relative uptake of32P by cassava,banana, elephant foot yam and groundnut in intercropping systems

Absorption of applied³²P by the treated as well as neighbouring plants in two- and three-crop intercropping systems involving cassava (Manihot esculenta Crantz), banana (Musa (AAB) Mysore), elephant foot yam (Amorphophallus campanulatus Blume) and groundnut (Arachis hypogaea L.) was studied in field trials. Radiophosphorus applied to the root zone of one of the component species in the mixed systems was found to be absorbed not only by the treated plant but also by the neighbouring plants. Banana was the most dominant species in the cassava-banana-elephant foot yam intercropping system and accumulated the major portion of the radioactivity recovered in the whole system. Cassava planted on raised mounds absorbed³²P from the root zones of elephant foot yam and banana growing in the interspaces. Absorption of³²P from cassava mounds by elephant foot yam was negligible.In cassava-groundnut intercropping system, cassava was the dominant component accumulating about 96 to 99 per cent of the total³²P recovery in the system when the radiolabel was applied to cassava and about 48 to 88 per cent when applied to the intercrops depending on whether cassava was planted on paired row-ridge, mound or flat bed. The groundnut was able to absorb only negligible quantity of³²P from cassava root zone. The absorption of³²P by treated groundnut was highest in paired-row ridge method of planting and lowest in flat bed method of planting.