A dual mechanism for intracellular pH regulation by leech neurones.

Schlue, Wolf-Rüdiger; Thomas, R C

doi:10.1113/jphysiol.1985.sp015748

Cited by 83 publications

(59 citation statements)

References 22 publications

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“…It is evident that the pHi returned to normal in both cases, and that its recovery was somewhat faster in C02-HCO3--buffered saline than in HEPES-buffered saline, which agrees with previous results (Schlue & Thomas, 1985). The membrane potential was between -47 mV and -52 mV during the course of the experiment, except during the exposure to 20 mM-NH4Cl, when it depolarized by 15 mV.…”

Section: Intracellular Ph Of Glial Cells and Neuronessupporting

confidence: 81%

“…The electrical arrangements were the same as described previously (Schlue & Deitmer, 1984;Schlue & Thomas, 1985). Each channel of a double-barrelled micro-electrode was connected to one input of a differential electrometer (WPI, F-223A) by chlorided wires.…”

Section: Micro-electrodesmentioning

confidence: 99%

“…Double-barrelled pH-sensitive micro-electrodes based on a neutral carrier sensor similar to those described recently (Schlue & Thomas, 1985) and for K+-sensitive micro-electrodes were made (Schlue & Deitmer, 1980 Fig. 1.…”

Section: Micro-electrodesmentioning

confidence: 99%

“…We have also measured the pHi of neurones in the same preparation and have compared the steady-state pHi and the regulation of pHi under various conditions in glial cells and neurones. The nervous system of the leech has appeared particularly suitable for this kind of study for the following reasons: (1) individual neurones and glial cells can be identified and penetrated by micro-electrodes (Nicholls & Kuffler, 1964; Baylor & Nicholls, 1969;Jansen & Nicholls, 1973; Schlue, Schliep & Walz, 1980;Walz & Schlue, 1982); and (2) several types of ion-sensitive micro-electrodes have been successfully used for extra-and intracellular recording of ions in the leech central nervous system (Schlue & Deitmer, 1980;Deitmer & Schlue, 1981Schlue & Wuttke, 1983;Schlue & Thomas, 1985). Our results reveal both similarities and differences concerning the maintenance of pHi in the two types of brain cells.…”

Section: Introductionmentioning

confidence: 99%

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The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

Deitmer

Schlue

1987

The Journal of Physiology

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135

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SUMMARY1. Double-barrelled, neutral-carrier pH-sensitive micro-electrodes were used to measure the intracellular pH (pHi) and the pHi regulation of neuropile glial (n.g.) cells and of identified neurones of the leech Hirudo medicinalis.2. The distribution of H+ in the n.g. cells and in the neurones was found not to be in electrical equilibrium. The mean pHi ofthe n.g. cells was 6-87 + 0 13 (mean + S.D.of mean here and for all following data n = 27) in HEPES-buffered leech saline (pHo = 7 4) and 7418 + 0-19 (n = 13) in 2 % C02-11 mM-HC03--saline. The mean pHi was 7'28+0-1 (n = 20) in Retzius neurones and 7-32+0415 (n = 12) in noxious neurones in HEPES-buffered leech saline, and 7-20+0415 (n = 10) and 7-27 +016 (n = 6) in 2 % C02-1 1 mM-HC03--buffered saline in these two types of neurones, respectively.3. The cytoplasmic buffering power, as calculated by the change in pHi following the change from 2 % C02-11 mM-HC03-to 5 % C02-22 mM-HCO.-in the leech saline, was 20-30 mM/pH unit in the n.g. cells and between 12 and 33 mM/pH unit in the neurones.4. The recovery of pHi in n.g. cells from an experimentally induced acidification (addition and removal of 20 mm-NH4Cl) was dependent on the presence of external Na+. Independent of the buffer system used, pHi recovery was inhibited when external Na+ was exchanged by N-methyl-D-glucamine. Amiloride (2-3 mM) reduced the rate of pHi recovery by about 50 % in these n.g. cells.5. In CO2-HCO3--free saline, or in the presence of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS, 0 5 mM), pHi recovery from an acid load was often slowed by up to 50 % in n.g. cells. This suggests that there is a significant contribution of a HC03--dependent membrane transport to pHi regulation in n.g. cells.6. When a HEPES-buffered saline was exchanged by a 2 % C02-11 mM-HCO3-buffered saline, the pHi of n.g. cells increased by 0-31 pH units. This alkaline shift was reversible upon removal of the CO2-HC03-and was absent in the Na+-free saline. It was not inhibited by 1 mM-furosemide or by 0 5 mM-SITS. Successive addition of first Na+ and then CO2-HCO3-to an Na+-free HEPES-buffered saline produced two separate phases of intracellular alkalization, suggesting two different pHi regulation transport systems.J. W. DEITMER AND W.-R. SCHLUE 7. In sensory and Retzius neurones pHi recovery from acidification was also dependent on external Na+, both in the presence and in the absence of CO2-HC03-.The rate of pHi recovery was reversibly reduced by 2 mM-amiloride by up to 90 % in these neurones.8. In conclusion, bicarbonate buffer affects n.g. cell pHi differently from the way in which it affects neuronal pHi. The regulation of pHi depends on the presence of external Na+ in both n.g. cells and neurones. The experiments suggest the presence of an Na+-H+ exchange and a SITS-sensitive HCO3--and Na+-dependent acid extrusion mechanism across n.g. (and nerve cell) membranes and another, SITSinsensitive HC03-and Na+-dependent transport system only in n.g. cells.

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Section: Intracellular Ph Of Glial Cells and Neuronessupporting

confidence: 81%

Section: Micro-electrodesmentioning

confidence: 99%

Section: Micro-electrodesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

Deitmer

Schlue

1987

The Journal of Physiology

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135

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“…3C). To alter intracellular pH without change of the pH of the extracellular solution we applied 20 mM NH 4 Cl, which is known to alkalize cell interior followed by acidification [14]. This was done to rule out the possibility, that ageladine A, which seems to be located in the cell membrane and the cytosol (Fig.…”

Section: Resultsmentioning

confidence: 99%

Ageladine A, a pyrrole–imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

Bickmeyer

Grube

Klings

et al. 2008

Biochemical and Biophysical Research Communications

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a b s t r a c tThe alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.Ó 2008 Elsevier Inc. All rights reserved.The pyrrole-imidazole alkaloid ageladine A was first isolated and described by Fujita et al.[1] using bioassay guided fractionation of extracts of the marine sponge Agelas nakamurai.The alkaloid showed biological effects such as the inhibition of matrix metallo-proteinases and the inhibition of cell migration of bovine endothelial cells. In 2006 the total synthesis of ageladine A was completed by the groups of Weinreb and Karuso [2,3] and later optimized [4,5].Brominated pyrrole-imidazole alkaloids are known to be fish feeding deterrent against the reef fish Thalassoma bifasciatum [6,7] and demonstrate antibiotic activity [8] even against pathogens like Helicobacter pylori [9]. Especially, the degree of bromination and the guanidine moieties have shown to be important for the alkaloids efficacy to disturb cellular calcium ion entry via voltage operated channels in neuroendocrine cells [10][11][12], which possess mainly L-and N-type calcium channels common in neuronal cells. During these fluorescence measurements of cellular effects by pyrrole-imidazole alkaloids, ageladine A was noticed to show fluorescence during UV excitation [12], which was also described earlier by Fujita et al. [1]. We demonstrate here other surprising properties of ageladine A such as its sensitivity to pH changes covering a wide range and because of its high membrane permeability an easy whole animal pH sensitive staining. Material and methodsCulture methods. PC12 cells from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were kept in culture medium containing RPMI 1640, 10% fetal calf serum, 5% horse serum, and 100 U penicillin/streptomycin per milliliter. Cells were cultivated in an incubator at 37°C, 90% humidity and 5% CO 2 with regular medium changes twice a week or when additionally necessary. Cells grew in culture flasks and 1-2 days prior to the experiments were seeded into petri dishes coated with coll...

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Ionic Mechanisms of Intracellular pH Regulation in the Nervous System

Schlue

Deitmer

2007

Ciba Foundation Symposium 139 ‐ Proton Passage Across Cell Membranes

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A dual mechanism for intracellular pH regulation by leech neurones.

Cited by 83 publications

References 22 publications

The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

Ageladine A, a pyrrole–imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

Ionic Mechanisms of Intracellular pH Regulation in the Nervous System

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