Showing papers by "Max Planck Society published in 1992"

Heteromeric NMDA receptors: Molecular and functional distinction of subtypes

Abstract: The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1) Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and NMDA-activated currents only when they were in heteromeric configurations with NR1 NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Abstract: Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.

Adsorbate-substrate and adsorbate-adsorbate interactions of Na and K adlayers on Al(111)

Abstract: We present total-energy, force, and electronic-structure calculations for Na and K adsorbed in various geometries on an Al(111) surface. The calculations apply density-functional theory together with the local-density approximation and the ab initio pseudopotential formalism. Two adsorbate meshes, namely, (\ensuremath{\surd}3 \ifmmode\times\else\texttimes\fi{} \ensuremath{\surd}3 )R30\ifmmode^\circ\else\textdegree\fi{} and (2\ifmmode\times\else\texttimes\fi{}2), are considered and for each of them the geometry of the adlayer relative to the substrate is varied over a wide range of possibilities. By total-energy minimization we determine stable and metastable geometries. For Na we find for both adsorbate meshes that the ordering of the calculated binding energies per adatom is such that the substitutional geometry, where each Na atom replaces a surface Al atom, is most favorable and the on-top position is most unfavorable. The (\ensuremath{\surd}3 \ifmmode\times\else\texttimes\fi{} \ensuremath{\surd}3 )R30\ifmmode^\circ\else\textdegree\fi{} structure has a lower energy than the (2\ifmmode\times\else\texttimes\fi{}2) structure. This is shown to be a substrate effect and not an effect of the adsorbate-adsorbate interaction. In contrast to the results for Na, we find for the (\ensuremath{\surd}3 \ifmmode\times\else\texttimes\fi{} \ensuremath{\surd}3 )R30\ifmmode^\circ\else\textdegree\fi{} K adsorption that the calculated adsorption energies for the on-top, threefold hollow, and substitutional sites are equal within the accuracy of our calculation, which is \ifmmode\pm\else\textpm\fi{}0.03 eV. The similarity of the energies of the on-surface adsorption sites is explained as a consequence of the bigger size of K which implies that the adatom experiences a rather small substrate electron-density corrugation. Therefore for potassium the on-top and hollow sites are close in energy already for the unrelaxed Al(111) substrate. Because the relaxation energy of the on-top site is larger than that of the threefold hollow site both sites receive practically the same adsorption energy. The unexpected possibility of surface-substitutional sites is explained as a consequence of the ionic nature of the bonding which, at higher coverages, can develop strongest when the adatom can dive into the substrate as deep as possible. The interesting result of the studied systems is that the difference in bond strengths between the ``normal'' and substitutional geometries is sufficiently large to kick out a surface Al atom.

Intersection theory on the moduli space of curves and the matrix Airy function

Abstract: We show that two natural approaches to quantum gravity coincide. This identity is nontrivial and relies on the equivalence of each approach to KdV equations. We also investigate related mathematical problems.

Depletion of intracellular calcium stores activates a calcium current in mast-cells

Abstract: In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.

The Structured Clinical Interview for DSM-III-R (SCID). II. Multisite test-retest reliability.

Abstract: A test-retest reliability study of the Structured Clinical Interview for DSM-III-R was conducted on 592 subjects in four patient and two nonpatient sites in this country as well as one patient site in Germany. For most of the major categories, kappa s for current and lifetime diagnoses in the patient samples were above .60, with an overall weighted kappa of .61 for current and .68 for lifetime diagnoses. For the nonpatients, however, agreement was considerably lower, with a mean kappa of .37 for current and .51 for lifetime diagnoses. These values for the patient and nonpatient samples are roughly comparable to those obtained with other structured diagnostic instruments. Sources of diagnostic disagreement, such as inadequate training of interviewers, information variance, and low base rates for many disorders, are discussed.

The distribution of 13 GABAA receptor subunit mRNAs in the rat brain − I. Telencephalon, diencephalon, mesencephalon

Abstract: The expression patterns of 13 GABAA receptor subunit encoding genes (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta) were determined in adult rat brain by in situ hybridization. Each mRNA displayed a unique distribution, ranging from ubiquitous (alpha 1 mRNA) to narrowly confined (alpha 6 mRNA was present only in cerebellar granule cells). Some neuronal populations coexpressed large numbers of subunit mRNAs, whereas in others only a few GABAA receptor-specific mRNAs were found. Neocortex, hippocampus, and caudate-putamen displayed complex expression patterns, and these areas probably contain a large diversity of GABAA receptors. In many areas, a consistent coexpression was observed for alpha 1 and beta 2 mRNAs, which often colocalized with gamma 2 mRNA. The alpha 1 beta 2 combination was abundant in olfactory bulb, globus pallidus, inferior colliculus, substantia nigra pars reticulata, globus pallidus, zona incerta, subthalamic nucleus, medial septum, and cerebellum. Colocalization was also apparent for the alpha 2 and beta 3 mRNAs, and these predominated in areas such as amygdala and hypothalamus. The alpha 3 mRNA occurred in layers V and VI of neocortex and in the reticular thalamic nucleus. In much of the forebrain, with the exception of hippocampal pyramidal cells, the alpha 4 and delta transcripts appeared to codistribute. In thalamic nuclei, the only abundant GABAA receptor mRNAs were those of alpha 1, alpha 4, beta 2, and delta. In the medial geniculate thalamic nucleus, alpha 1, alpha 4, beta 2, delta, and gamma 3 mRNAs were the principal GABAA receptor transcripts. The alpha 5 and beta 1 mRNAs generally colocalized and may encode predominantly hippocampal forms of the GABAA receptor. These anatomical observations support the hypothesis that alpha 1 beta 2 gamma 2 receptors are responsible for benzodiazepine I (BZ I) binding, whereas receptors containing alpha 2, alpha 3, and alpha 5 contribute to subtypes of the BZ II site. Based on significant mismatches between alpha 4/delta and gamma mRNAs, we suggest that in vivo, the alpha 4 subunit contributes to GABAA receptors that lack BZ modulation.

Cloning and expression of a rat brain L-glutamate transporter.

Abstract: SYNAPTIC transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia1–5. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels5,6. Here we use an antibody against a glial L-glutamate transporter from rat brain7 to isolate a complemen-tary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8–9 putative transmembrane α-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation

Abstract: Vascular endothelial growth factor (VEGF) is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. Such factors are likely candidates for regulatory molecules involved in endothelial growth control. We have characterized the murine VEGF gene and have analysed its expression pattern in embryogenesis, particularly during brain angiogenesis. Analysis of cDNA clones predicted the existence of three molecular forms of VEGF which differ in size due to heterogeneity at the carboxy terminus of the protein. The predicted mature proteins consist of 120, 164 or 188 amino acid residues. Homodimers of the two lower molecular weight forms, but not of the higher molecular weight form, were secreted by COS cells transfected with the corresponding cDNAs and were equally potent in stimulating the growth of endothelial cells. During brain development, VEGF transcript levels were abundant in the ventricular neuroectoderm of embryonic and postnatal brain when endothelial cells proliferate rapidly but were reduced in the adult when endothelial cell proliferation has ceased. The temporal and spatial expression of VEGF is consistent with the hypothesis that VEGF is synthesized and released by the ventricular neuroectoderm and may induce the ingrowth of capillaries from the perineural vascular plexus. In addition to the transient expression during brain development, a persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in choroid plexus and in kidney glomeruli. The data are consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation.

Correction for liquid junction potentials in patch clamp experiments.

Abstract: This chapter describes corrections that have to be applied to measured membrane potentials in patch clamp experiments. Some of them [Eqs. (1)-(3)] are required regardless of the nature of the reference electrode (in the Ringer's solution bath) whenever the pipette-filling solution is different from the bath solution. They represent the liquid junction potentials that are present at the pipette tip before patch formation. In addition, corrections have to be applied when the bath solution is being changed during a measurement (i.e., after seal formation). In that case the following rules apply. (1) The new solution should never get into contact with the bare silver/silver chloride wire of the reference electrode. This requirement is best met by using a salt bridge. (2) The "best" salt bridge is a 3 M KCl bridge with an abrupt KCl-bath fluid boundary at its tip (see above). This bridge does not require any additional potential corrections, but it may lead to KCl poisoning of the bath or become contaminated by solutions used previously. (3) Local solution changes (microperfusion by puffer pipette, U tool or sewer pipe arrangements) as well as recessed KCl bridges require additional corrections, which (together with the simple liquid junction potential correction) are approximately given by Eqs. (6)-(8). It should be stressed that all equations given here represent approximate corrections, since liquid junction potentials are thermodynamically ill-defined. This is particularly relevant for Eqs. (6) and (7) where the sum of two liquid junction potentials appears.

Natural protein proteinase inhibitors and their interaction with proteinases

Abstract: Proteinase inhibitors are important tools of nature for regulating the proteolytic activity of their target proteinases, for blocking these in emergency cases, or for signaling receptor interactions or clearance Endogenous inhibitors appear to be always proteins; small non-proteinaceous inhibitors which impair the proteolytic activity of host proteinases are produced in microorganisms

Tethered chains in polymer microstructures

Abstract: Tethered polymer chains refers to macromolecular chains that are attached into microstructures by their ends. Highly branched polymers, polymer micelles and end-grafted chains on surfaces are a few examples. This review brings out the common features of these seemingly widely disparate microstructures. Tethering can be reversible or irreversible and is frequently sufficiently dense that the chains are crowded. Densely tethered chains stretch to alleviate the interactions caused by crowding. They thus exhibit deformed configurations at equilibrium. These effects of tethering on the structure of the polymer chains are reflected in distinctive behavior and properties of microstructures containing tethered chains.

Dewetting of thin polymer films

Abstract: Thin polystyrene films (100 nm) on silicon substrates undergo dewetting when annealed above the glass transition temperature. Three different stages can be distinguished: The smooth films break up by the creation of cylindrical holes. The holes then grow and form rims ahead of them which finally contact each other creating ``cellular'' structures. The rims are unstable and decay into droplets. The influence of the film thickness on this process is investigated and compared to recent theoretical predictions of spinodal decomposition of partially wetting thin films.

Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter.

Abstract: Transcription of the proto-oncogene c-fos is stimulated rapidly and transiently by serum growth factors and mitogens. Critical for this response is the serum-response element which is bound in vivo in a ternary complex containing the transcription factors p67SRF and p62TCF (ref. 2). Disruption of the ternary complex correlates with impaired induction by serum and phorbol ester. Mitogen-activated protein (MAP) kinase is a serine/threonine kinase which is activated 1-5 minutes after treatment of cells with mitogens and growth factors that induce re-entry into the cell cycle, making MAP kinase a candidate for the transmission of proliferative signals. Here we show that p62TCF is phosphorylated by MAP kinase in vitro and that phosphorylation results in enhanced ternary complex formation. Serum-starved Swiss 3T3 cells treated with epidermal growth factor, which induces MAP kinase in these cells, are induced to express c-fos and yield p62TCF active in ternary complex formation. In contrast, treatment of Swiss 3T3 cells with insulin, which does not activate MAP kinase under these conditions, does not lead to enhanced ternary complex formation nor does it induce c-fos transcription. Our results link the expression of the human c-fos proto-oncogene to signal transduction pathways known to be activated before its own induction.

Synaptotagmin: a calcium sensor on the synaptic vesicle surface

Abstract: Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic vesicles. However, the molecular steps transducing the calcium signal into membrane fusion are still an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle protein, binds calcium at physiological concentrations in a complex with negatively charged phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of synaptotagmin (it is abolished by proteolytic cleavage at a single site). These results suggest that synaptotagmin acts as a cooperative calcium receptor in exocytosis.

Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway

Abstract: The mesolimbic dopaminergic system has been implicated in mediating the motivational effects of opioids and other drugs of abuse. The site of action of opioids within this system and the role of endogenous opioid peptides in modulating dopamine activity therein remain unknown. Employing the technique of in vivo microdialysis and the administration of highly selective opioid ligands, the present study demonstrates the existence of tonically active and functionally opposing mu and kappa opioid systems that regulate dopamine release in the nucleus accumbens, the major terminal area of A10 dopaminergic neurons. Thus, stimulation of mu-type receptors in the ventral tegmental area, the site of origin of A10 dopaminergic neurons, increases dopamine release whereas the selective blockade of this opioid receptor type results in a significant decrease in basal dopamine release. In contrast, stimulation of kappa-type receptors within the nucleus accumbens decreases dopamine release whereas their selective blockade markedly increases basal dopamine release. These data show that tonic activation of mu and kappa receptors is required for the maintenance of basal dopamine release in the nucleus accumbens. In view of the postulated role of the mesolimbic system in the mediation of drug-induced alterations in mood and affect, such findings may have implications for the treatment of opiate dependence and affective disorders.

Electron Localization in Solid-State Structures of the Elements: the Diamond Structure

Abstract: verify this result half quantitatively using a model kit as analog computer. The different sizes of C and Si are simulated with tetrahedral joints whose arm lengths differ[*] and the atoms are joined by flexible bonds (bent bonds). In disilabicyclo[l .1 .O]butane C,Si,H, (2) the region between the two C atoms does have a high ELF value (Fig. 1 c and 1 d). This confirms the previously described bond.['] The relatively small region of high ELF values implies a weak bond, in agreement with the long bond length. The white ELF maximum is also clearly off the straight topological CC connecting line. Its position is remarkably close to that of the bent bond derived from the simple structural model.IZ1 As expected, there is no bond between the Si atoms (Fig. 1 d). on the Cray-2 in Stuttgart. Mr. M. Kohout (Universitat Stuttgart) contributed to the development of the program MEROP (for the calculation of the electron density and of ELF) and wrote the program MPLOT (for drawing the contour lines of Fig. 2). The methods for obtaining localized orbitals-often used in the chemistry of molecules to describe bonding-can be used in principle for solids as well (in methane and in diamond , for example). They can lead, however, to several equivalent sets of orbitals for a given structure and are non-unique in this case. This ambiguity occurs, for example, in monomeric monocycles such as benzene, or in an infinite polyene chain.\"] In solids ambiguity often arises on account of the higher coordination, and localized orbitals are therefore used only rarely. An analysis in positional space can nevertheless be performed when instead of the equivocal localized orbitals, the electron localization function (ELF) is used. In this work we have calculated ELF for crystalline solids for the first time. The electron localization function was introduced by Becke and Edgecombe as a measure of the probability of finding an electron in the neighborhood of another electron with the same spin.\"] ELF is thus a measure of the Pauli repulsion. The explicit formulation is given in Equation (a) The parameter K is the curvature of the electron pair density for electrons of identical spin, e(r) the density at (Y), and Kh the value of K in a homogeneous electron gas with density e. The ELF values lie by definition between zero and one. Values are close to 1 when in the vicinity of one …

Bursty bulk flows in the inner central plasma sheet: An effective means of earthward transport in the magnetotail

Abstract: High speed flows in the Earth's Inner Central Plasma Sheet (ICPS) occur during enhanced flow intervals that have been termed Bursty Bulk Flow (BBF) events. The importance of different flow magnitude samples for Earthward transport in the ICPS are statistically evaluated and several representative BBF's and their relevance to Earthward transport are discussed. The selection of BBF's is automated in a database and they are shown to be responsible for most of the Earthward transport that occurs within the ICPS. The BBF related transport is compared to the transport measured within the entire plasma sheet during the 1985 AMPTE/IRM crossings of the magnetotail. The results show that BBF's last only a small fraction of the time in the plasma sheet but can account for several tens of percent of the Earthward particle and energy transfer and possibly all of the Earthward magnetic flux transfer in the plasma sheet.

New plant binary vectors with selectable markers located proximal to the left T-DNA border

Abstract: Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.

Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells

Abstract: In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.

Fluorescence resonance energy transfer and nucleic acids.

Abstract: Publisher Summary Fluorescence resonance energy transfer (FRET) is a spectroscopic process by which energy is passed nonradiatively among molecules over long distances. The “donor” molecule, which must be a fluorophore, absorbs a photon and transfer this energy nonradiatively to the “acceptor” molecule. Energy can be transferred over distances on the order of common macromolecular dimensions. This chapter discusses the applications of FRET to nucleic acid molecules conjugated to dye molecules; but in general, the donor-acceptor pairs can be free in solution, one or both bound to a macromolecule, or be an inherent part of the structure. The chapter discusses FRET specifying the physical parameters that are available from a variety of fluorescence measurements. In the chapter, some basic considerations and experimental procedures are described that are necessary to make reliable FRET measurements. It also discusses some difficulties that can arise in the experimental proceedings and data analysis and the sources of experimental error are pointed out.

Calcium channel characteristics conferred on the sodium channel by single mutations.

Abstract: THE sodium channel, one of the family of structurally homologous voltage-gated ion channels1, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin2. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance2. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which ccurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in deter mining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.

Calcium gradients and buffers in bovine chromaffin cells

Abstract: 1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.