Simultaneous measurement of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography/mass spectrometry

Jenden, Donald J.; Roch, Margareth; Booth, Ruth A.

doi:10.1016/0003-2697(73)90134-6

Cited by 220 publications

(52 citation statements)

References 13 publications

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“…Subsequently, consecutive 2 h incubations were made in 1 ml eserine-frog Ringer solution aliquots for biochemical assay. At the conclusion of the experiment, all aliquots and homogenized nerve-muscle preparations were assayed for ACh content by gas chromatography/mass spectrometry as previously described (Jenden, Roch & Booth, 1973).…”

Section: Chemical Measurements Of Ach Effluxmentioning

confidence: 99%

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.

Grinnell¹,

Gundersen²,

Meriney³

et al. 1989

The Journal of Physiology

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SUMMARY1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside-out clamped patches ofXenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve-evoked patch currents (EPCs) and spontaneous quantal 'miniature' patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones.2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4-1 pmol (2 h)-1, and no significant difference in resting efflux between enzyme-treated and control preparations. The ratio of enzymetreated to contralateral control muscle efflux averaged 1-17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1-7 ,UM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67 % of control.3. In the absence of nerve stimulation, the frequency of single-channel openings recorded by outside-out patch probes adjacent to nerve terminals was very low (1-2 min-'), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non-quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches.4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non-quantally from the synaptic surface of the frog nerve terminal is less than one-tenth the amount expected if all non-quantal release is from this region of the terminal membrane.5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single-channel openings increased for several seconds. This transient increase in channel openings was not sensitive to movement of the t To whom correspondence should be sent. A. D. GRINNELL AND OTHERS patch electrode a significant distance (4 /tm) away from the active sites, or to manipulations previously reported to block non-quantal transmitter leakage, including addition of 10 mM-Ca2+ or 1-7 fM-vesamicol to the bath. These channel openings appear to be due to an accumulation of ACh which originated from many evoked quanta, and not the effect of locally increased non-quantal ACh release due to nerve stimulation.6. We conclude that transmitter leakage at adult frog terminals is either localized to a source other than the synaptic surface of the nerve terminal, or released in a widespread and diffuse fashion from many sources, which may include the nerve terminal.

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Section: Chemical Measurements Of Ach Effluxmentioning

confidence: 99%

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.

Grinnell¹,

Gundersen²,

Meriney³

et al. 1989

The Journal of Physiology

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“…The tissue was weighed and then extracted overnight as described previously (Gundersen, Jenden & Newton, 1980). A GCMS assay was used to measure ACh and choline in all samples (Jenden, Roch & Booth, 1973;Freeman Choi & Jenden, 1975;Gundersen et al, 1980 The rate of choline output from control preparations ranged from 1.5 to 2.6 pmol mg-1 min 1, while the output after oxotremorine (10 or 100 gM) was 1.4 to 2.3 pmol mg-' min-. Similarly, tissue choline content of controls averaged 56 + 9 pmol/mg, while those exposed to oxotremorine (10 pM) contained 61 ± 7 pmol/mg.…”

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confidence: 99%

Oxotremorine Does Not Enhance Acetylcholine Release From Rat Diaphragm Preparations

Gundersen

Jenden

1980

British J Pharmacology

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We have reinvestigated the dramatic effect of oxotremorine on acetylcholine release from the rat diaphragm reported by Das, Ganguly & Vedasiromoni (1978), using a rigorous gas chromatographic mass spectrometric/isotope dilution method for identification and measurement of acetylcholine and choline. Oxotremorine (10 μm) causes no significant change in the spontaneous or evoked (1 or 10 Hz) release or in the tissue levels of acetylcholine or choline.

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“…A volatile derivative of choline (demethylation with benzenethiolate and formation of propionyl ester) was then purified using gas chromatography (6 ftx 2 mm i.d. glass column packed with 3% OV-17 on GC22 precoated with 1% 4-dodecyldiethylenetriamine succinamide) and measured with a mass selective detector (MSD; Hewlett Packard 5970 GC/MSD) [12]. Use of internal standards allowed correction for recovery of losses during the isolation procedures.…”

Section: Methodsmentioning

confidence: 99%

1,2‐sn‐Diacylglycerol accumulates in choline‐deficient liver

Blusztajn¹,

Zeisel²

1989

FEBS Letters

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Choline deficiency is associated with triacyiglycerol accumulation in the liver, and is the only nutritional state known to trigger hepatic cancer spontaneously. In two different experiments, rats were pair-fed for 6 weeks with control (0.2% choline), or choline-deficient (CD) (0.002% choline) diets. Hepatic choline and phosphocholine declined in CD animals to 54% and 16~ of control levels, respectively. In control livers, 1,2-sn-diacylglycerol (I,2-sn-DAG) concentration was (in nmol/g wet wt) 144 (+25; mean+SE); while in CD livers it was 792 (+ 140) in the first experiment. In the second experiment the values were 375 (+ 26) and 1147 (+ 74), respectively. 1,2-sn-DAG, a precursor of triacylglycerol, is an endogenous activator of protein kinase C (PKC). PKC is the presumed site of action of the tumor-promoting phorbol esters. We suggest that the 1,2-sn-DAG accumulating in CD liver could bind PKC, altering its activity, and thus contribute to the carcinogenic effect of CD diets.

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Simultaneous measurement of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography/mass spectrometry

Cited by 220 publications

References 13 publications

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.

Oxotremorine Does Not Enhance Acetylcholine Release From Rat Diaphragm Preparations

1,2‐sn‐Diacylglycerol accumulates in choline‐deficient liver

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