Alvarez-Leefmans, Francisco J.

Boyarsky, G.

Marquez, S.

Rosas, Vania D.

English

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Wright State University CORE Scholar Pharmacology and Toxicology Faculty Publications Pharmacology and Toxicology 1999 Quantitative Analysis of Changes in Cell Volume and Intracellular pH Induced by Ammonia and Ammonium in Neuroblastoma Cells Studied with Fluorescent Probes Vania D. Rosas G. Boyarsky University of Texas S. Marquez Francisco J. Alvarez-Leefmans Wright State University - Main Campus, francisco.alvarez-leefmans@wright.edu Follow this and additional works at: https://corescholar.libraries.wright.edu/ptox  Part of the Chemicals and Drugs Commons Repository Citation Rosas, V. D., Boyarsky, G., Marquez, S., & Alvarez-Leefmans, F. J. (1999). Quantitative Analysis of Changes in Cell Volume and Intracellular pH Induced by Ammonia and Ammonium in Neuroblastoma Cells Studied with Fluorescent Probes. Journal of General Physiology, 114 (1), 8a-9a. https://corescholar.libraries.wright.edu/ptox/13 This Conference Proceeding is brought to you for free and open access by the Pharmacology and Toxicology at CORE Scholar. It has been accepted for inclusion in Pharmacology and Toxicology Faculty Publications by an authorized administrator of CORE Scholar. For more information, please contact library-corescholar@wright.edu. 8a the journal of general physiology ⋅ volume 114 ⋅ 1999system employs custom-made quartz AODs (positioning time,10 ms; IntraAction) and is coupled with a UV-reflecting di-chroic mirror to an inverted microscope fitted with a UV-trans-mitting objective lens (Fluar 1003, N.A. 1.3; Zeiss). Concurrentacousto-optical modulation (AOM) of beam intensity is used tocontrol the pulse energy on a point-by-point basis and thus allowsfor graded photolysis. A monolayer of cultured hippocampalneurons was used to test this system. Bath-applied cagedglutamate (200 mM, CMB-g cage; Molecular Probes) was photo-lyzed with single laser pulses (z5 mJ at preparation), and recep-tor currents were measured in whole-cell voltage clamp duringblockage of transmitter release with cadmium (50 mM). AMPAand NMDA components were differentiated pharmacologically(25 mM APV and 10 mM CNQX). No response to multiple pho-tolysis pulses was detected in the presence of both antagonists.Receptor currents could be graded by modulating the pulse en-ergy over a restricted range. Future extensions include combin-ing this micro-photolysis system with Random-access Fluores-cence Microscopy to form a powerful tool for simultaneous opti-cal stimulation and recording. (Supported in part by NationalScience Foundation grant IBN-9723871 to P. Saggau.)28. Characterization of Quinoline Sulfonamide Zn-sensitiveFluorescent Probes for Use in Live Tissues  VLADISLAVSNITSAREV and ALAN R. KAY, Department of Biological Sci-ences, University of Iowa, Iowa City, Iowa (Sponsor: T. Hoshi)Ionic Zn is found at high concentrations (.100 mM) in vesi-cles in some cells, notably in the terminals of some glutamatergicterminals in the mammalian forebrain. Two water-soluble fluo-rescent probes, Zinquin and TFLZn, have recently been devel-oped based on the Zn21 sensitivity of the quinoline sulfonamidemoiety that have proved useful in studying vesicular Zn. We havecharacterized the interactions of these probes with Zn21 in mem-branous systems using unilamellar liposomes. We have focusedthis study on Zinquin, which was found to have a higher quan-tum yield than TFLZn. Zinquin-free acid was found to crossmembranes passively, obviating the need for an acetoxy methyl(AM) ester derivative for loading. The stoichiometry of the com-plexes formed between Zn21 and Zinquin and the location ofthese complexes in liposomal solutions was studied by a combi-nation of fluorescence spectroscopy and fluorescence lifetimemeasurements. In aqueous solution, the 1:2 [M/L] complex wasfavored. In liposomes, both 1:1 and 1:2 complexes were formed,both partitioning into the membrane, with the 1:2 complex ableto act as an ionophore. The ionophoretic activity was blocked bya transliposomal pH gradient of z2 U similar to that foundacross synaptic vesicles.In live tissue, Zn21 probes may diffuse across membranes pas-sively or be actively taken up by a transporter or endocytosis. Todistinguish between these pathways, the rate of Zinquin uptakewas measured as a function of temperature, as below 158C, en-docytosis is blocked and active transport would be more severelyretarded than passive transport. The uptake of Zinquin was mea-sured in rat neocortical slices held in a constantly circulating oxy-genated saline at 26 and 88C. The time course of loading at bothtemperatures appeared biexponential, with slower rates at lowertemperature. The Q10 for both processes was z1.5, which is con-sistent with diffusion accounting entirely for the movement ofZinquin. 9Supported by NIH grant NS-35243).29. Cellular Distribution, Protein Interactions, and Ag-gregate Formation of Huntingtin's Amino Terminus UsingGFP Variants and Fluorescence Resonance Energy Trans-fer (FRET) Analysis SHAWN D. HANDRAN, MARCY E.MACDONALD, and ANNE B. YOUNG, Department of Neu-rology and Molecular Neurogenetics Unit, Massachusetts GeneralHospital, Boston, Massachusetts (Sponsor: C. Gonzalez)Huntingtin is a large protein of unknown function and con-tains a polyglutamine segment near the NH2 terminus. DNA in-stability and expansion of the CAG tract that encodes the poly-glutamine stretch results in Huntington's Disease (HD). The cel-lular dynamics of huntingtin and its putative interacting proteinswere investigated by generating fusion proteins using the cyan(ECFP) and yellow (EYFP) variants of green fluorescent protein(GFP). EYFP was fused in-frame with truncated huntingtin (aa1–550, termed HDN) containing normal (Q32) or mutant(Q100) polyglutamine segments. ECFP was fused in-frame tofragments of three different huntingtin yeast partners (HYP;Faber et al. 1998. Hum. Mol. Genet. 7:1463–1474): HYP A-ECFP,HYP B-ECFP, and HYP L-ECFP. COS cells from confluent cul-tures were singly or doubly transfected by electroporation withHDN Q32 or Q100-EYFP and/or HYP A, B, or L-ECFP constructs.Cellular distribution of HDN was largely cytoplasmic, with littleor no nuclear staining. Some transfected cells exhibited HDN ag-gregates, which were also observed in cells transfected with HDNcontaining nonpathogenic range of glutamine repeats (Q32).Expression of HYP A-ECFP (a fragment of FBP11, a WW do-main–containing splicosome protein) alone appeared to be toxicto cells, as abnormal morphological appearance was observedover a period of several days after transfection. Cellular localiza-tion was nuclear and cytoplasmic. HYP B-ECFP, a WW domain–containing protein fragment of unknown function, was localizedalmost exclusively to the nucleus, with very little or no detectablecytoplasmic signal. HYP L-ECFP, a fragment of human FIP-2 pro-tein containing a leucine zipper domain, was diffusely localizedthroughout the cytoplasm and nucleus. Preliminary results coex-pressing HYP B-ECFP and HDN Q32 or Q100-EYFP show thatthere is almost no colocalization, and to date, redistribution ofeither has not been robustly observed, although some instancesof altered HYP B-ECFP distribution were seen. HYP L-ECFP andHDN-EYFP have extensive overlap in the cytoplasm, but notwithin the nucleus. Interestingly, there is recruitment of HYPL-ECFP into HDN aggregates in cells exhibiting aggregate forma-tion. Preliminary observation of FRET between HDN-EYFP andHYP B-ECFP was negative, perhaps due to the lack of cellularcolocalization. Testing of FRET in additional pairings is ongoing,as is generation of full length protein constructs. ECFP was a giftof Dr. R.Y. Tsien. (Supported by the Hereditary Disease Founda-tion and NIH grants AG-13617 and NS-32765.)30. Quantitative Analysis of Changes in Cell Volume andIntracellular pH Induced by Ammonia and Ammonium inNeuroblastoma Cells Studied with Fluorescent ProbesVANIA D. ROSAS,‡ G. BOYARSKY,i S. MARQUEZ,‡§ andF.J. ALVAREZ-LEEFMANS,*‡§ *Department of Pharmacology,Cinvestav, IPN, Apartado Postal 14740, México D.F., Mèxico,‡Department of Neurobiology, IMP, Av. México-Xochimilco 101México D.F., México, §Department of Physiology and Biophysics,Wright State University, Dayton, Ohio, and  iUniversity of TexasMedical Branch, Galveston, Texas53rd Annual Meeting ⋅ Society of General Physiologists 9aElevated levels of ammonia (NH3) and ammonium (NH41)lead to neuronal and glial swelling and may contribute to thebrain edema seen in various hepatic diseases. The Pathophysiol-ogy of this edema is not well understood. Moreover, exposure ofcells to NH3/NH41 is a commonly used experimental maneuveraimed at displacing intracellular pH (pHi) in a controlled andpredictable manner to study its regulation and yet the volumechanges associated with this maneuver are not well understood.Here, we show a method whereby simultaneous changes in cellwater volume (CWV) and pHi can be studied quantitatively in sin-gle cells. CWV and pHi were measured simultaneously in singleneuroblastoma cells (N1E-115 and NG108) loaded with BCECF.pHi was measured ratiometrically, and CWV was determined atthe isosbestic wavelength (440 nm). Independent CWV measure-ments were done using calcein, a fluorescent probe insensitive tointracellular ions (Alvarez-Leefmans et al. 1995. Methods Neurosci.27:361). Experiments were done with solutions buffered withHepes without added bicarbonate. Upon exposure to 20-mMNH4Cl isosmotic solutions, the cells swelled by 12.7 6 1.7%above their initial CWV in 1.5 6 0.1 min (n 5 8) and then dis-played isosmotic regulatory volume decrease (RVD). Upon re-moval of NH3/NH41, the cells shrank and recovered their initialvolumes with a variable time course. Simultaneously recordedchanges in pHi  suggest that most of the swelling may be due tointracellular NH41 accumulation. The latter would result fromprotonation of intracellular NH3 and by direct entry of NH41 viachannels or carriers. The recovery of the shrinkage after NH3/NH41 removal could be blocked by amiloride (1 mM), suggest-ing the involvement of Na1/H1 exchange in isosmotic regula-tory volume increase. (Supported by NINDS grant no. 29227 anda Guggenheim Fellowship to F.J. Alvarez-Leefmans.)31. Designing Photoregulated Ion Channels and En-zymes ANDREW WOOLLEY, A. JAMES, D. BURNS, A.GOVINDARAJAN, D. LIU, L. LIEN, and J. ZHANG, De-partment of Chemistry, University of Toronto, Toronto, Canada(Sponsor: B.M. Salzberg)Photoregulated artificial ion channels would enable direct ma-nipulation of cellular excitability using light. Photoregulated en-zymes could be tools for dissecting complex cellular chemistry.We have prepared a first-generation photomodulated ion chan-nel that is a derivative of the peptide gramicidin. This derivativehas a COOH-terminal extension that is designed to act as a pho-togate at the entrance (and exit) of the channel. We have pre-pared a series of ribonuclease S analogues with photoisomeriz-able residues at various unique positions in the enzyme structure.Different types of photoisomerizable amino acids based on anazobenzene core have been synthesized: phenyl-azophenylala-nine (Pap); 4-carboxy-phenyl-azophenylalanine (C-Pap), and aconstrained derivative, phenylazo-1,2,3,4-tetrahydro-3-isoquinoli-necarboxylic acid (Patic). The effects of these moieties on en-zyme activity can be rationalized in structural terms using dataderived for NMR and molecular modeling of the modified en-zymes. The design strategy, modeled structures and observed ac-tivities of these analogues will be discussed.32. Use of Optical Probes to Study Presynaptic Regula-tion of Monoamine Quantal Size in CNS Neurons andSecretory Cells EMMANUEL N. POTHOS, VIVIANADAVILA, and DAVID SULZER, Departments of Neurologyand Psychiatry, Columbia University, and Department of Neuro-science, New York State Psychiatric Institute, New YorkRecently, amperometric recordings have provided the meansto detect presynaptic quantal release of monoamine transmittersfrom axonal varicosities and cell bodies (Wightman et al. 1991.PNAS. 88:10754; Zhou and Misler. 1995. Biophys. J. 39A:398;Pothos et al. 1998. J. Neurosci. 18:5575; Jaffe et al. 1998. J. Neurosci.18:3548). We adapted these approaches to record quantaldopamine release from small synaptic vesicles in axonal varicosi-ties of CNS neurons (Pothos et al. 1998. J. Neurosci. 18:4106).As this approach records only transmitter released directly un-der the electrode, the use of markers to identify active presynap-tic sites may improve data collection. We used FM1-43 (4 mM) tolabel presynaptic sites in midbrain dopamine neuronal culturesbefore and after exposure to secretagogues (40 mM K1, 20 nMa-latrotoxin). Neurons are initially maintained in Ca21-free me-dia for 10 min, showing little decrement in FM1-43 label. If sub-sequent application of secretagogue decreases FM1-43 label at asite, we find that it is often a good candidate for amperometricrecording. We have also used nicotine as a more selective secreta-gogue, as nicotinic receptors are highly expressed at dopaminepresynaptic terminals. This axonal terminal localization of nico-tine receptors was confirmed by fluorescent a-bungarotoxin (Or-egon Green), which labels dopaminergic terminals in the livingpreparation with fairly good specificity, as seen by subsequent fix-ation and immunostain for tyrosine hydroxylase.In amperometric recordings from cultured adrenal chromaf-fin cells, we examined the effects of stimulation on quantal size.We find that depolarization increases uptake of the fluorescentweak base vital dye acridine orange (AO) in chromaffin granules.Interestingly, both vesicular acidification and quantal size wereincreased by the nicotinic agonist DMPP (10 mM). Both effectswere blocked by the removal of extracellular Ca21, the protonpump inhibitor bafilomycin, and the Cl2 channel blocker NPPB.The combination of AO fluorescence and amperometry suggeststhat vesicular acidification, which may depend on increased Ca21entry, increases quantal size. This effect may be due to potentia-tion of a vesicular Cl2 current.33. Calcium Release Channels are Localized to Neuro-secretory Granules in Terminals of the Mouse Neuro-hypophysis B.M. SALZBERG, M. MUSCHOL, S.D.KRANER, and A.L. OBAID, Departments of Neuroscience andPhysiology, University of Pennsylvania School of Medicine, Phila-delphia, PennsylvaniaThe neurosecretory terminals of the vertebrate neurohypo-physis have long provided an important model system for investi-gation of the mechanisms that mediate the rapid release of neu-rotransmitters and neuropeptides. From the hypothalamus (su-praoptic and paraventricular nuclei in mammals; preopticnucleus in amphibia), magnocellular neurons project their ax-ons as bundles of fibers through the median eminence and in-fundibular stalk, to arborize extensively and terminate in theneurohypophysis, where the neuropeptides vasopressin and oxy-tocin are released into the circulation by a calcium-dependentmechanism. In the rat, some 2 3 104 neurons give rise to z4 3107 neurosecretory terminals and swellings, whose membranesaccount for nearly 99% of the excitable membrane in the neuro-hypophysis (pars nervosa). The dense packing of terminals prob- Published July 1, 1999

Quantitative Analysis of Changes in Cell Volume and Intracellular pH Induced by Ammonia and Ammonium in Neuroblastoma Cells Studied with Fluorescent Probes

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Quantitative Analysis of Changes in Cell Volume and Intracellular pH Induced by Ammonia and Ammonium in Neuroblastoma Cells Studied with Fluorescent Probes

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