Diffusion of acetylcholine in agar gels and in the isolated rat diaphragm

Krnjević, K.; Mitchell, Jude F.

doi:10.1113/jphysiol.1960.sp006555

Cited by 62 publications

(15 citation statements)

References 16 publications

(16 reference statements)

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“…The diffusion rate constant is somewhat smaller than that for free diffusion [given as 6-10 x 10-6 cm2 S-1 (28)(29)(30)]. …”

Section: Discussionmentioning

confidence: 99%

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

Land¹,

Harris²,

Salpeter³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the fallins phase of the MEPC. We find diffusion to be 4 x 10-6 cm2 slightly slower than free diffusion, forward binding to be 3.3 x 107 M-s-, and the distance from an average release site to the nearest exit from the cleft to be 1.6 jtm. We also estimate the back reaction rates. Heidmann and Changeux (7).] In our approach we use the shape of the MEPC as a function of AcChoR concentration to derive forward binding rates as well as information on AcCho diffusion in the intact neuromuscular cleft. We thus complement the results obtained by chemical techniques and by single-channel recording. The derived parameters support our motivating assumptions of the "saturating disk model" used by Salpeter and colleagues (8-11) to explain the generation of the MEPC. METHODSWe recorded MEPCs by voltage clamp from the intercostal muscle of the lizard Anolis carolinensis, using experimental techniques as in the first two papers of this series (1, 2) with minor modifications.Three groups were used: (i) control muscle (esterases and receptor concentration unaltered); (ii) receptors intact and esterases fully inactivated [with 1 mM diisopropyl fluorophosphate (iPr2P-F) for 1 hr]; (iii) receptors partially inactivated [with 40 nM a-bungarotoxin (BTX) for 40 min] to -0.36 its normal value (papers 1 and 2) and esterases inactivated with iPr2P-F as in ii. The filtering conditions of the voltage clamp were modified from those used in papers 1 and 2 to improve the signal-to-noise ratio. Because the falling phase of MEPCs is slow relative to the rise time, the signals were low-pass-filtered at 1 kHz (two-pole Butterworth filter) and the digitizer sampling rate was reduced to 6.25 kHz. Even though the Nyquist frequency is only a factor of 3 above our filter cutoff, aliasing was judged to be less than 4% of peak current. All experiments were done at 22 ± 10C and -100 mV holding potential. Fall time (tf) values were obtained from the experimental MEPC as 1.23 x the 90-40% fall time. The factor 1.23 is the conversion from 90-40% to efolding time for a pure exponential. Fig. 1 shows six examples of measured MEPC traces, illustrating the three conditions used in the present study. Fig. 2 plots the relation between tf and MEPC amplitude Ac, and Table 1 gives the mean values of tf and Ac under these conditions. RESULTSWe confirm (12-15) that inactivation of esterases lengthens tf appreciably and that, when cr is reduced by BTX treatment, tf is somewhat shortened again (12, 15). Our additional observations are that, when esterases are intact, tf does not depend on Ac ( Fig. 2A), but after esterase inactivation tf increases with increasing Ac (Fig. 2B). Finally, decreasing a, with esterases inactivated, alm...

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“…The diffusion rate constant is somewhat smaller than that for free diffusion [given as 6-10 x 10-6 cm2 S-1 (28)(29)(30)]. …”

Section: Discussionmentioning

confidence: 99%

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

Land¹,

Harris²,

Salpeter³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Unfortunately, none of these three parameters is precisely known. If ACh moved in the cleft by free diffusion (Do 10-5 cm2/sec-1; Fatt, 1954;Eccles & Jaeger, 1958;Krnjevic &Mitchell, 1960, andDryer & it would spread over the critical area of 03 #,m2 in < 15 ,usec. However, as suggested by Gage & McBurney (1975) Kd values are generally given as < 4 x 10-6 M (Mautner, Bartels & Webb, 1966;Dolly & Barnard, 1974;Meunier, Sealock, Olsen & Changeux, 1974;Moreau & Changeux, 1976).…”

Section: Discussionmentioning

confidence: 99%

Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications.

Matthews-Bellinger¹,

Salpeter²

1978

The Journal of Physiology

214

124

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SUMMARY1. The distribution of acetylcholine receptors (AChR) at frog cutaneous pectoris neuromuscular junctions was studied quantitatively using [125I]a-bungarotoxin (a-BTX) labelling and EM autoradiography.2. We found that, as in mouse end-plates, the AChR is localized uniformly along the thickened post-junctional membrane. In the frog muscle this specialized membrane constitutes approximately the top 50 % of the junctional folds.3. The receptor site density is 26,000 + 6000 sites/,um2 on the thickened postjunctional membrane and falls sharply to 50 sites/fum2 within 15 Itm from the axon terminal.4. a-BTX site density on the presynaptic axonal membrane was directly determined to be at most 5 % of the value on the thickened post-junctional membrane.5. The high post junctional AChR site density leads us to conclude that: (a) each quantum of ACh needs to spread only over a very small post-junctional area (to be called the 'critical area') before it encounters as many AChR (plus AChE) sites as there are ACh molecules in the quantum (for a packet of 104 ACh molecules this critical area is-03 #um2), (b) the average concentration of ACh prevailing in

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“…It has been shown that the obstruction effect and charge on the agar gel have an insignificant influence on diffusion (Krnjevi6 & Mitchell, 1960;Lauffer, 1961;Schantz & Lauffer, 1962;Winn, Smith, Campbell & Huf, 1964;Slade, Cremers & Thomas, 1966). Analysis procedures At the conclusion of the experiment, the electrode slope, m, interference, K, and system constant, R. were determined from electrode calibration in 8itu, as described above.…”

Section: Ionophore8ismentioning

confidence: 99%

Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.

Nicholson¹,

Phillips²

1981

The Journal of Physiology

752

685

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SUMMARY1. The validity of the macroscopic laws of ion diffusion was critically examined within the microenvironment of the extracellular space in the rat cerebellum using ion-selective micropipettes and ionophoretic point sources.2. The concepts of volume averaging, volume fraction (a) and tortuosity (A) were defined and shown to be theoretically appropriate for quantifying diffusion in a complex medium such as the brain.3. Diffusion studies were made with the cations tetramethylammonium and tetraethylammonium and the anions a-naphthalene sulphonate and hexafluoroarsenate, all of which remained essentially extracellular during the measurements. Diffusion parameters were measured for a period of 50 s and over distances of the order of 0-1 mm. 4. Measurements ofthe diffusion coefficients of the ions in agar gel gave values that were very close to those derivable from the literature, thus confirming the validity of the method.5. Measurements in the cerebellum did not reveal any systematic influences of ionophoretic current strength, electrode separation, anisotropy, inhomogeneity, charge discrimination or uptake, within the limits tested. 6. The pooled data from measurements with all the ions gave a = 0-21 +002 (mean+s.E. of mean) and A = 1-55+0-05 (mean+S.E. of mean).7. These results show that the extracellular space occupies about 20 % of the rat cerebellum and that the diffusion coefficient for small monovalent extracellular ions is reduced by a factor of 2-4 (i.e. A2) without regard to charge sign. The over-all effect of this is to increase the apparent strength of any ionic source in the cerebellum by a factor of A2/a, about 12-fold in the present case, and to modify the time course of diffusion.8. These conclusions confirm that the laws of macroscopic diffusion are closely obeyed in the cerebellum for small ions in the extracellular space, provided that volume fraction and tortuosity are explicitly taken into account. It is likely that these conclusions are generally applicable to other brain regions and other diffusing substances. PHY 321C. NICHOLSON AND J. M. PHILLIPS

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Diffusion of acetylcholine in agar gels and in the isolated rat diaphragm

Cited by 62 publications

References 16 publications

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications.

Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.

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