microM Choline Research Articles

Simultaneous Determination of Glucose and Choline Based on the Intrinsic Fluorescence of the Enzymes

It has been possible to perform the simultaneous determination of choline and glucose using the intrinsic fluorescence of the corresponding enzyme as an analytical signal. This can be done in two ways. First, for low glucose and choline concentrations (about 0.55 mM and 0.75 microM respectively) two differentiated signals, without mutual interference, are obtained for both analytes in the same measurement. Second, when glucose and choline concentrations are higher, a new model has been designed which permits the concentrations to be accurately determined in samples containing from 0.55 mM to 3.75 mM glucose and from 0.75 microM to 11.0 microM choline; the method has been applied to simultaneous glucose and choline determinations in serum samples with good results. This method gives a better performance than multivariate calibration based on Partial Least Squares Regression. The methodology here shown could be also used for the simultaneous determination of other pairs of analytes.

Effect of Acetylcholine Precursors on Proliferation and Differentiation of Astroglial Cells in Primary Cultures

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5'-diphosphocholine (CDP-choline) and alpha-glyceryl-phosphorylcholine (alpha-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 microM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 microM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 microM alpha-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 microM choline or alpha-GPC, whereas in 24 h 1 microM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 microM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 microM alpha-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.

Carrier-Mediated Transport of the Quaternary Ammonium Neuronal Nicotinic Receptor Antagonist N,N′-Dodecylbispicolinium Dibromide at the Blood-Brain Barrier

The quaternary ammonium compound N,N'-dodecyl-bispicolinium dibromide (bPiDDB) potently and selectively inhibits nicotinic receptors (nAChRs) mediating nicotine-evoked [(3)H]dopamine release and decreases nicotine self-administration, suggesting that this polar, charged molecule penetrates the blood-brain barrier (BBB). This report focuses on 1) BBB penetration of bPiDDB; 2) the mechanism of permeation; and 3) comparison of bPiDDB to the cations choline and N-octylnicotinium iodide (NONI), both of which are polar, charged molecules that undergo facilitated BBB transport. The BBB permeation of [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB was evaluated using in situ rat brain perfusion methods. Cerebrovascular permeability surface-area product (PS) values for [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB were comparable (1.33 +/- 0.1, 1.64 +/- 0.15, and 1.3 +/- 0.3 ml/s/g, respectively). To ascertain whether penetration was saturable, unlabeled substrate was added to the perfusion fluid. Unlabeled choline (500 microM) reduced the PS of [(3)H]choline to 0.15 +/- 0.06 microl/s/g (p < 0.01). Likewise, unlabeled bPiDDB (500 microM) reduced the PS of [(14)C]bPiDDB to 0.046 +/- 0.005 microl/s/g (p < 0.01), whereas unlabeled NONI reduced the PS for [(3)H]NONI by approximately 50% to 0.73 +/- 0.31 microl/s/g. The PS of [(14)C]bPiDDB was reduced (p < 0.05) in the presence of 500 microM choline, indicating that the BBB choline transporter may be responsible for the transport of bPiDDB into brain. Saturable kinetic parameters for [(14)C]bPiDDB were similar to those for [(3)H]choline. The current results suggest that bPiDDB uses the BBB choline transporter for approximately 90% of its permeation into brain, and they demonstrate the carrier-mediated BBB penetration of a novel bisquaternary ammonium nAChR antagonist.

Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells

Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

Characterization of human erythrocyte choline transport in chronic renal failure.

Membrane transport of choline cations is elevated in renal failure in erythrocytes and cerebral tissue but the origins and clinical importance of this are unknown. The membrane transport changes have been characterized using erythrocytes from patients on maintenance haemodialysis (HD), patients on continuous ambulatory peritoneal dialysis (CAPD), and control subjects. Data were obtained from cells depleted of intracellular choline to create zero-trans (ZT) conditions for choline influx. [14C]-choline influx measurements provided a kinetic description of choline flux as the sum of a saturable transport system (defined by Vmax and Km) and an apparent diffusion pathway. Inhibition of choline transport by hemicholinium-3 (HC-3), quinine and N-ethylmaleimide (NEM) has been studied. Actions of three cationic polyamine putative uraemic toxins (putrescine, spermidine, spermine) were tested in control erythrocytes. Mean (SEM) Vmax (ZT) was increased in HD at 45.0 (3.0) mumol/l cells/h and in CAPD at 46.6 (2.5) mumol/l cells/h compared to controls (30.0 (2.0) mumol/l cells/h). Mean Km (ZT) was not significantly altered in HD or CAPD (HD: 6.1 (1.6) microM; CAPD: 5.5 (0.7) microM; control: 5.1 (0.9) microM). The sensitivity of choline transport to the inhibitors tested was not altered in HD. 1.0 mM quinine, 2.0 mM NEM and 1.0 mM HC-3 caused 75-90% inhibition of transport in both HD and controls. For inhibition of ZT influx of 25 microM choline the mean IC50 of quinine was 90 (9) microM in HD and 101 (13) microM in controls (n.s.). The ZT influx of 200 microM choline was not altered by any of the polyamines at concentrations up to 1.0 mM. Membrane choline transport in CRF remains protein-mediated and exhibits normal substrate and inhibitor affinities; high values of Vmax seem to occur through increased surface expression of an active normal choline transporter. Increases in plasma polyamines cannot explain the choline transport changes in CRF.

Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes.

The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.

Phosphatidylcholine biosynthesis is required for secretion of truncated apolipoprotein Bs from McArdle RH7777 cells only when a neutral lipid core is formed

Decreased phosphatidylcholine biosynthesis inhibits the secretion of very low density lipoproteins from hepatocytes (Yao, Z. and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004). We have now investigated whether or not phosphatidylcholine biosynthesis is required for secretion of carboxyl-truncated apoBs 15, 18, 23, 28, and 37 from transfected McArdle RH7777 cells. Transfected cells were choline-deprived for 1 day and then choline-supplemented or maintained choline-deficient. Pulse-chase experiments showed that in the presence of 100 microM choline, the secretion of apoBs 28 and 37 was increased by up to 50 and 45%, respectively, whereas the secretion of apoBs 15, 18, and 23 was not affected by the addition of choline. Immunoblot analyses also showed that choline in the medium increased the secretion of apoBs 28 and 37 by 30-50% whereas the secretion of apoBs 15, 18, and 23 was unaffected over 24 h. As only carboxyl-terminal truncations with a size greater than 23% of apoB-100 are able to assemble a neutral lipid core (McLeod, R. S., Y. Zhao, S. L. Selby, J. Westerlund, and Z. Yao. 1994. J. Biol. Chem. 269: 2852-2862), we conclude that only apoB-truncations that assemble a neutral lipid core require phosphatidylcholine synthesis. Other experiments established that the requirement of phosphatidylcholine for apoB secretion is related to the presence of neutral lipid associated with a truncation, rather than the length of apoB.

Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/2 fibroblasts.

We have investigated the possible requirement of phosphatidylcholine for normal progression through the cell cycle of C3H/10T1/2 fibroblasts. Incubation of the cells in a medium with 0.5% serum synchronized the cells in the G0 stage of the cell cycle. Supplementation of the cells with 10% dialyzed and delipidated serum +/- choline resulted in normal cell division and growth for cells with choline, whereas cell division was markedly impaired in the absence of choline. Flow cytofluorometry analysis indicated that after 4 days in the absence of choline, 85% of the fibroblasts were in the G1 phase. Addition of choline resulted in synchronous synthesis of DNA with a peak occurring after 14 h. Incubation of cells with 0.5% serum had no effect on phosphatidylcholine (PC) levels in cells supplemented with 28 microM choline, but the concentration of PC was reduced from 32 to 20 nmol/10(6) cells after 1 day of incubation in the absence of choline. Supplementation with dialyzed serum, but not dialyzed and delipidated serum, allowed choline-deficient cells to replicate normally. This was attributed to the presence of lysophosphatidylcholine in dialyzed serum as this lipid, but not other lipids (e.g., phosphatidylcholine or mitogenic lipids) was able to replace the choline requirement. The choline-deficient effect was not complete; some DNA synthesis occurred in the absence of choline in the medium, and approximately 30% of the cells completed mitosis in 35 h compared to 100% in the presence of choline. The data suggest that phosphatidylcholine is required for normal progression of the cell cycle beyond the G1 phase and is unrelated to the induction of G0 to G1 transition. Choline deficiency should be a useful method for synchronizing cells in the G1 phase.

Quantitation of Choline in the Extracellular Fluid of Brain Tissue with Amperometric Microsensors

Amperometric microsensors for the detection of choline in the extracellular fluid of brain tissue have been prepared by immobilizing horseradish peroxidase and choline oxidase onto carbon fiber microcylinder electrodes with a cross-linkable redox polymer. The microcylinders have diameters of 7 or 10 microns and lengths of 200-400 microns. To detect choline, the microsensors are operated at an applied potential of -0.1 V vs SCE. At this potential, ascorbate and other easily oxidizable interferant molecules present in brain tissue are not detected by the electrode. Ascorbate, however, can interfere with the response to choline by acting as a reducing agent in the enzyme-containing polymer film. So, a Nafion overlayer is required in order to reliably detect choline in the presence of physiologically relevant concentrations of ascorbate (approximately 200 microM). The Nafion-coated microsensors have a detection limit of approximately 5 microM choline and give a linear response beyond 100 microM when calibrated in vitro at 37 degrees C. Exposure of the microsensors to brain tissue for several hours causes less than a 10% loss in redox polymer surface coverage and less than a 25% loss in sensitivity to choline. To assess the ability of the microsensors to monitor choline levels in brain tissue, small volumes of a choline solution were injected into brain tissue at a site about 1 mm away from a microsensor. The current arising at the microsensor was converted to choline concentration by calibrating the sensor following the in vivo experiment. The resultant choline concentrations were in excellent agreement with those predicted by appropriate diffusion equations.

Incorporation of [3H]ethanolamine into acetylcholine by a human cholinergic neuroblastoma clone

Human neuroblastoma cholinergic LA-N-2 cells were used as an experimental model to test the possibility that the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) and free choline (Cho) (Andriamampandry et al. 1989) could contribute to acetylcholine (AcCho) synthesis. LA-N-2 cells were incubated with [3H]Cho for 90 min and 22.7% of the radioactivity was present in PCho, 18.5% in free Cho and 4.8% as AcCho. The ratio of Cho/AcCho, however, was of about 1 after 16 hours of incubation. The incorporation of 10 microM [3H]ethanolamine (Etn) into MeEtn, PMeEtn, PMe2Etn and their corresponding phospholipids was reduced in cells incubated in medium containing 7.2 microM choline as compared to cells incubated in medium devoid of choline indicating that the lack of Cho from the incubation medium stimulated the conversion of PEtn to Cho water soluble derivatives. Incubation of LA-N-2 cells with [3H]Etn led to the labelling of [3H]AcCho. Cultures incubated in parallel with [3H]Cho showed that roughly 10% of [3H]AcCho obtained after 16 hrs of incubation with the Cho label derived from [3H]Etn. The synthesis of Cho and AcCho from Etn may be enhanced after cellular differentiation induced by the growth of the cells in the presence of retinoic acid (RA). The results indicate that the methylation of [3H]Etn and/or of [3H]PEtn may be used by cholinergic neurons as precursor for AcCho.

Transport of choline by plasma membrane vesicles from lung-derived epithelial cells.

A549 cells, a lung epithelium-derived cell line, were used as a model system to study choline transport by granular pneumocytes. Intact cells accumulated free choline against a concentration gradient by a low-affinity transport system with kinetic characteristics similar to that previously described for granular pneumocytes (Am. J. Respir. Cell Mol. Biol. 1: 455, 1989). Membrane vesicles prepared from these cells showed a 10-fold enrichment in plasma membrane marker enzymes with a vesicular H2O space of 5.7 +/- 0.05 (SE) microliters/mg protein. Vesicles showed a time- and concentration-dependent uptake of free [3H]choline in Na(+)-free medium. With 5 microM choline, choline uptake reached an apparent steady-state concentration gradient (inside/outside) of 50. 3H that was membrane associated ("bound" choline) represented approximately 5% of total uptake. In the presence of an initial gradient of NaCl, choline uptake showed an overshoot with a plateau value similar to Na(+)-free conditions; a similar effect was observed for plasma membrane vesicles from rat lung type 2 epithelial cells. The steady-state uptake of choline was inhibited at low pH (6.5) and by the presence of valinomycin or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone and was abolished when both were present. These results show that plasma membrane vesicles from A549 cells accumulate choline by binding to the membranes and by Na(+)-dependent and -independent transport mechanisms, the latter apparently reflecting a transmembrane proton gradient.

A choline transporter in renal brush-border membrane vesicles: energetics and structural specificity.

Choline is a quaternary ammonium compound that is normally reabsorbed by the renal proximal tubule, despite its acknowledged role as a substrate for the renal organic cation (OC) secretory pathway. The basis for choline reabsorption was examined in studies of transport in rabbit renal brush-border membrane vesicles (BBMV). Although an outwardly directed H+ gradient (pH 6.0in: 7.5out) stimulated uptake of tetraethylammonium (TEA), a model substrate of the OC/H+ exchanger in renal BBMV, it had no effect on uptake of 1 microM choline. A 5 mM trans concentration gradient of choline did, however, drive countertransport of both TEA and choline, although trans TEA had no effect on choline accumulation in BBMV. A 20 mM concentration of unlabeled choline blocked uptake of both choline and TEA by greater than 85%, whereas 20 mM TEA blocked only TEA uptake. The kinetics of choline uptake into vesicles preloaded with 1 mM unlabeled choline appeared to involve two, saturable transport processes, one of high affinity for choline (Kt of 97 microM) and a second of low affinity (Kt of approximately 10 mM), the latter presumably reflecting a weak interaction of choline with the OC/H+ exchanger. An inside-negative electrical PD stimulated the rate of uptake and supported the transient concentrative accumulation of choline in BBMV. The high affinity transporter showed a marked specificity for choline and closely related analogues. A model of the molecular determinants of substrate-transporter interaction is described. We conclude that the electrogenic high affinity pathway plays a central role in renal reabsorption of choline.

The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 microM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KCl. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 microM choline did not lead to a further increase, but preincubation with 25 or 50 microM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 microM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 mM KCl, ACh release from slices preincubated with 50 microM choline was greater than from slices preincubated with 0, 10, or 25 microM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KCl, the ACh content of slices preincubated with 0 or 10 microM choline decreased significantly, whereas that of slices preincubated with 25 or 50 microM choline did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.

Demonstration of a Choline Requirement for Optimal Keratinocyte Growth in a Defined Culture Medium

Nutrient requirements for proliferation and differentiated function of individual cell types can be determined using cell culture methodologies. Human epidermal keratinocytes are stimulated to grow by choline supplementation in the presence of myo-inositol when grown in a commercial nutrient medium containing six other defined supplements. The optimal range of choline concentrations varied among donor cell lines, but consistently fell between 36 microM and 180 microM. Addition of 72 microM choline increased cell yield to 250 +/- 38% of that produced by myo-inositol supplementation alone and 92 +/- 8% of that produced by addition of a highly mitogenic hypothalamic extract, which was previously required for good growth in this culture system. Supplementation of the basal medium with both the extract and choline resulted in 165 +/- 13% of the cell yield observed with the extract addition alone. Supplementation with other phospholipid precursors did not further increase keratinocyte growth. Neither dermal fibroblasts nor epidermal melanocytes were stimulated by supplementation with choline, suggesting the high keratinocyte requirement is unusual. This completely defined culture medium for keratinocyte growth should prove useful in analyzing the role of phospholipids and other nutrients in human epidermis.

Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration.

Nominally K-sensitive microelectrodes were used to measure carbachol (CCh) in order to study the dependence of muscarinic effects on CCh concentration and exposure time in guinea pig hippocampal slices. Interference presumably originating from tissue choline-compounds was neutralized by pre-equilibration of the slices with 500 microM choline and calibration of the CCh-sensitive microelectrodes in the presence of the same choline-concentration. Muscarinic depolarization and reduction of the afterhyperpolarization (AHP) following a train of action potentials by bath applied CCh were monitored in granule cells and CA3 pyramidal neurons by intracellular recording. A fast bath application mode of CCh was designed, by which CCh tissue concentration reached a peak after 2-3 min and was washed out with a half time of about 8 min. After application of 30 nmol CCh in this way, the AHP was reduced according to the variation of CCh concentration over time. Neurons depolarized with some delay after the reduction of the AHP and started to repolarize 1 min before the peak of tissue CCh concentration (0.6 microM) was reached. Pirenzepine (1-10 microM) blocked only the depolarization, while atropine (1-10 microM) blocked both the depolarization and the reduction of the AHP. When superfusing with CCh containing saline, 80% of the final concentration was reached in the bath after 12 min, but in the tissue only after 45 min. The slow increase of tissue CCh concentration was concurrent with the slow decrease of the AHP. No effect on the membrane potential was observed. Atropine, but not pirenzepine, blocked the reduction of the AHP. Superfusion with a high CCh concentration (100-300 microM) containing saline depolarized neurons and reduced the AHP. Then pirenzepine repolarized neurons, whereas atropine both repolarized the cells and restored the AHP. It is concluded tha the muscarinic depolarization depends not only on the CCh concentration, but also on the rate of rise of CCh, while the reduction of the AHP depends solely on the concentration. This result is discussed in terms of the possibility that the depolarization is mediated by a short term desensitizing M1 muscarinic receptor subtype and the reduction of the AHP is mediated by a M2 muscarinic receptor subtype.

Phosphatidylcholine as the choline donor in sphingomyelin synthesis.

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [methyl-14C]choline resulted in rapid accumulation of essentially all the label (approximately 95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 microM choline), loss of label from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 microM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 microM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9-12 hr until it reached the specific radioactivity of PC. In the presence of 300 microM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism.

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.

Acetylcholine turnover in an autoactive molluscan neuron.

We have studied acetylcholine (ACh) turnover at the cholinergic synapse between an identified motoneuron, the salivary burster (SB), and the muscle cells of the salivary duct (SD) in the terrestrial mollusk Limax maximus. Electrophysiological recordings were made of the SB action potentials and the SB-elicited junction potentials (JPs) on the SD. The amplitude of the JP was used as a measure of ACh release by the SB. The SB is an autoactive neuron that discharges 1 to 12 bursts of action potentials per min. During sustained bursting activity, the SB is able to maintain transmitter release for 18 hr even in the absence of exogenous choline. The size of SB-elicited JPs does not vary during 18 hr of activity. If the choline uptake blocker, hemicholinium-3 (HC-3; 20 microM), is present in the saline, transmitter release and JP size are depressed by about 30% after 14 hr of activity. Thus, the SB is partially dependent upon choline reuptake for maintained ACh synthesis and release. In high (9.45 mM)-potassium (K+) saline, the SB fired tonically at twice its average spike frequency. JP amplitude initially increased, then declined to an amplitude which was 60% of the initial level. The addition of 20 microM HC-3 to the high-K+ saline caused a 75 to 100% decrease in JP size within 30 min. Thus, during high-frequency tonic firing, the SB was primarily dependent on choline reuptake for ACh synthesis and release. After JP size had been reduced in high-K+ saline containing HC-3, the SB-SD synapse was returned to normal choline-free saline. The SB resumed bursting activity. JP amplitude gradually increased over the next 30 min. Thus, high-frequency firing in HC-3 had not depleted the SB of its entire endogenous store of choline or ACh. If the synapse was fatigued in high-K+ saline containing HC-3 and then placed in saline enriched with 300 microM choline, JP size increased within minutes. Thus, uptake of choline for ACh synthesis and release may be a more rapid process than mobilization of an endogenous transmitter store. Finally, the SB-SD synapse was fatigued in high-K+ saline containing HC-3. HC-3 was then removed from the saline. The SB maintained high-frequency tonic activity. JP size did not increase unless choline was added to the saline.(ABSTRACT TRUNCATED AT 400 WORDS)

Exogenous choline augments transmission at an identified cholinergic synapse in terrestrial mollusk Limax maximus.

1. A variety of pharmacological tests indicate that the neuromuscular junction between the salivary burster neuron (SB) and the salivary duct muscle (SD) in the terrestrial mollusk Limax maximus is cholinergic. These include the effects of curare, atropine, and hexamethonium. 2. Exogenously applied choline can act both presynaptically and postsynaptically at the synapse between the SB and SD. a) When the SB-SD synapse is bathed in 30 microM choline, there is no measurable direct postsynaptic effect at the SB-SD synapse. After several hours of choline incubation, however, an increase is observed in the size of SB-elicited junction potentials recorded from the SD. b) When the synapse is bathed in 300 microM choline, a short-term decrease is seen in the amplitudes of junction potentials (JPs) recorded from the SD. This effect is interpreted as competition between choline and acetylcholine (ACh) for postsynaptic receptor sites on the SD, since the response of the denervated SD to ACh is diminished in the presence of 300 microM choline. c) After several hours incubation in 300 microM choline, the amplitudes of JPs elicited by the SB on the SD increase. 3. The long-term effect of exogenous choline is blocked by the choline-uptake inhibitor, hemicholinium 3. 4. We conclude that exogenous choline is taken up by the presynaptic terminals of the SB and converted to ACh. Transmitter output rises with growing transmitter stores, producing an increase in the size of SB-elicited JPs recorded from the SD.