UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Calcium homeostatic mechanisms operating in cultured post-natal rat hippocampal neurons Sidky, Adam Omar 1997

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
ubc_1997-0477.pdf [ 4.22MB ]
[if-you-see-this-DO-NOT-CLICK]
Metadata
JSON: 1.0087958.json
JSON-LD: 1.0087958+ld.json
RDF/XML (Pretty): 1.0087958.xml
RDF/JSON: 1.0087958+rdf.json
Turtle: 1.0087958+rdf-turtle.txt
N-Triples: 1.0087958+rdf-ntriples.txt
Original Record: 1.0087958 +original-record.json
Full Text
1.0087958.txt
Citation
1.0087958.ris

Full Text

CALCIUM HOMEOSTATIC MECHANISMS OPERATING IN CULTURED POST-NATAL RAT HIPPOCAMPAL NEURONS by ADAM OMAR SIDKY B.Sc. (Biochemistry), The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept this thesis as confonping to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1997 © Adam Omar Sidky, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood, that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of W^lOU^ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) 11 ABSTRACT In neurons, calcium (Ca2+) is used for a variety of processes and, as a result, its intracellular regulation is an important factor in determining which messenger systems are activated and the extent of that activation. To date two principal methods have been used to induce increases in the free intracellular Ca2+ concentration ([Ca ];) in populations of neurons. These are the activation of either voltage or ligand gated Ca2+-channels by supervisions with either high K+-solutions or solutions containing ligand gated Ca2+-channel agonists. Unfortunately, with the use of either of these methods, substantial Ca2+-gradients are formed and decay kinetics are difficult to measure as a result of the lag time between agonist application and peak [Ca2+];. In order to circumvent these problems a novel method has been developed to examine potential Ca2+-homeostatic mechanisms in cultured post-natal rat hippocampal neurons. The method requires the monitoring of the recovery of background subtracted fluorescence levels of the Ca2+-indicator dye fluo-3 at 20-22 °C immediately following a rapid increase in [Ca2+]j induced by flash photolysis of the caged Ca2+-compound nitrophenyl-EGTA (NP-EGTA). A variety of methods or drugs were used in an attempt to block efflux of Ca2+ by the plasma membrane Na+/Ca2+ exchanger (PM-Na+/Ca2+) or uptake of Ca2+ into mitochondria. We found that many of the experimental manipulations produced a decrease in intracellular pH (pH;) measured in sister cultures using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). These changes in pH; are likely to influence neuronal Ca2+-homeostatic mechanisms directly by decreasing mitochondrial Ca2+-uptake, decreasing the affinity of Ca2+-binding proteins for Ca2+, and inhibition of the PM-ATPase. Accordingly, for each experimental situation we determined the appropriate amount of the weak base trimethylamine (TMA) required to restore baseline pH; prior to flash photolysis. Ill When the Na /Ca exchanger was inhibited by replacement of all extracellular Na with Af-methyl-D-glucamine (NMDG) we observed a significant prolongation in the rate of recovery to baseline Ca2+-levels. However, this treatment markedly reduced pH; and when this effect was corrected with 5 mM TMA, the resulting recovery rates of fluo-3 fluorescent intensities were virtually identical to those seen in control situations. Similar results were found when all external Na+ was replaced by Li+. These experiments were particularly revealing since the effects of Li+ on pH; were time dependent. At early time intervals pH; was reduced and there was an apparent reduction in the rate of recovery of fluo-3 fluorescent intensities. At later times pH; was restored towards normal values and no effect of blockade of the Na /Ca exchanger on Ca2+ recovery rates was observed. It is concluded therefore that, in our neuronal preparations, the Na+/Ca2+ exchanger is relatively unimportant in the removal of Ca2+-loads induced by the caged Ca2+-compound NP-EGTA. Inhibition of mitochondrial Ca2+-uptake, using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), resulted in a reduction in pH; which could be restored with 2 mM TMA. Under these conditions the rate of recovery of Ca2+-levels was significantly slower than controls. Similar results were found using the respiratory chain inhibitor rotenone. In order to avoid the potentially confounding effects that mitochondrial Ca2+-uptake inhibitors such as CCCP have on ATP-levels, oligomycin was added to the superfusate to block reverse activity of the ATP-synthase in order to sustain ATP levels, over the short term. Under these conditions we still observed a significant prolongation in the rates of recovery to baseline values. We conclude therefore that the uptake of Ca2+ into mitochondria is an important homeostatic mechanism in cultured post-natal rat hippocampal neurons following a Ca2+-load induced by photolysis of NP-EGTA. iv TABLE OF CONTENTS Page Abstract ii Table of Contents v List of Tables vList of Figures vii Acknowledgments ix INTRODUCTION Overview of neuronal Ca2+-homeostasis 1 Calcium entry into neurons 2 Voltage gated calcium channels 3 Ligand gated calcium channels 4 Intracellular calcium stores 5 Intracellular calcium homeostasis 6 Calcium ATPasesPlasma Membrane ATPase 7 Endoplasmic Reticulum ATPase 8 PM-Na7Ca2+ exchanger 9 Mitochondria 10 Calcium binding proteins 12 Overview of calcium homeostatic methods 13 Experimental methods for increasing[Ca2+]; 4 Effects of pH on Ca2+-homeostatic mechanisms 16 Goals of this study 18 MATERIALS AND METHODS Tissue Culture 19 Ca2+-imaging with fluo-3 and the use of NP-EGTA 19 Loading 1Imaging 20 NP-EGTA data analysis 22 Ca2+-imaging using fura-2 3 LoadingFura-2 data analysis 4 V Ca +-imaging using fura-red 25 Loading 2Fura-red data analysis 6 pH imaging using BCECF 7 LoadingBCECF data analysis 2RESULTS Loading conditions for fluo-3 and NP-EGTA 29 Neuronal calcium response to NMD A and caged calcium compound 30 Comparison of Room Temperature and 37 °C rates of recovery .... 46 Role of the PM-Na+/Ca2+ exchanger 49 Role of Mitochondria 54 Role of the Endoplasmic Reticulum ATPase 68 Summary and statistical analysis of data 81 DISCUSSION Loading with fluo-3 and NP-EGTA 85 NP-EGTA as a method for increasing [Ca2+]; 87 Rates of recovery of [Ca2+]; at 22° and 37° C 93 The influence of pH on Ca2+-homeostatic mechansims 94 The role of the PM-Na+/Ca2+ exchanger 6 The role of mitochondria 97 The role of the Endoplasmic Reticulum 101 Conclusions, limiatations and future directions 10REFERENCES 104 vi LIST OF TABLES Page Table 1 Loading parameters investigated for fluo-3-AM and NP-EGTA-AM 29 Table 2 Comparison of Room Temperature vs. 37 °C rates of recovery 49 Table 3 Statistical analysis of potential calcium homeostatic mechanisms 82 vii LIST OF FIGURES Page Figure 1 Typical control response in a field of neurons to NMDA application and flash photolysis 32 Figure 2 Typical response in a field of neurons to NMDA followed by two consecutive uncages 34 Figure 3 The influence of pH on NP-EGTA 36 Figure 4 The effects of the caged Ca2+-compound nitr-5 39 Figure 5 Response seen to 0.4 s flash in fura-2 and NP-EGTA loaded neurons ... 41 Figure 6 Response seen to 0.5 s flash in fura-2 and NP-EGTA loaded neurons ... 43 Figure 7 Response seen to a field of neurons loaded with fura-red and NP-EGTA .. 45 Figure 8 Typical response seen at 37 °C 48 Figure 9 The effects of 0Na+-NMDG on pFL 51 Figure 10 The effects of 0Na+-Li+on pH; 3 Figure 11 Background subtracted normalized data showing the influence of the PM-Na7Ca2+ exchanger on Ca2+-homeostasis 56 Figure 12 The effects of CCCP on resting pFL 8 Figure 13 The effects of CCCP and CCCP + TMA on pH; : 61 Figure 14 The effects of CCCP on resting Ca2+-levels 63 viii Page Figure 15 The effects of CCCP and CCCP + TMA on Ca2+-levels 66 Figure 16 Background subtracted normalized data showing the influence of CCCP on Ca2+-homeostasis 67 Figure 17 The effect of Rotenone on pH; 70 Figure 18 The effect of rotenone on Ca2+-levels 71 Figure 19 Background subtracted normalized data showing the influence of rotenone on Ca2+-homeostasis 74 Figure 20 The effect of thapsigargin on pH; 76 Figure 21 The effect of CPA on pH; 78 Figure 22 The effect of Ryanodine on pH; 80 Figure 23 The effect of clamping pHj using high K+-nigericin during NMDA application 84 IX ACKNOWLEDGMENTS First and foremost I would like to thank Dr. Kenneth G. Baimbridge for his guidance over these past three years. As a result of his care and concern for his students, my past three years have been an altogether enjoyable experience and will be looked back upon fondly. His support of both my academic and extracurricular endeavours was immeasurable and I truly appreciate his patience and understanding of my numerous absences. Secondly, I would like to thank Dr. John Church for his invaluable advice and expertise on numerous aspects of pH and Ca2+ imaging. His suggestions and advice were indispensible and helped formulate the backbone for many aspects of my project. I would also like to thank Stella Atmadja for her technical expertise. Without her proficiency, none of this work would have been possible. I would like to thank Dr. Edwin Moore and Dr. Steven Kehl who provided advice and helpful discussion on data analysis and a variety of technical aspects of my work along with the members of my supervisory committee who provided invaluable insights and suggestions. I would also like to thank the students and faculty of the Department of Physiology who have made the past three years extrordinarily enjoyable, and in particular, Dr. Raymond Pederson who makes graduate work in physiology an endless grad-retreat. 1 INTRODUCTION I. Overview of neuronal Ca2+-homeostasis Mechanisms designed to regulate intracellular calcium ion concentration ([Ca2+]j) are pivotal in the control of a wide variety of cellular functions. In particular, neurons are known to use calcium ions (Ca2+) as a trigger for diverse functions such as neurotransmitter release (Augustine et al, 1987), the opening of potassium channels and the control of neuronal membrane excitability (Morita et al, 1982), the induction of intracellular Ca2+-release (Fabiato, 1983), the control of membrane permeability (Meech, 1978), the induction of long term potentiation (Lynch et al, 1983) and the modulation of enzyme or gene activity (e.g. phospholipases, protein kinases, and endonucleases; Morgan and Curran, 1988). The mechanisms of Ca2+ entry, its subsequent short-term buffering and longer-term clearance, along with the mechanisms which affect the amplitude, shape and duration of the cytosolic Ca2+-signal in neurons are therefore important factors in determining which messenger systems are activated and the extent of that activation (Benham et al, 1992; Stuenkel, 1994). In addition, prolonged increases in [Ca2+]; may lead to cell death (Randall and Thayer, 1992) and Ca2+-signals intended to produce a particular physiological response (Werth et al, 1996) may, if uncontrolled, have a similar result. Therefore a balance exists between the [Ca2+]; needed in order to induce rapid Ca2+ signaling versus the [Ca2+]j at which point toxicity becomes a factor. As a result, [Ca2+]; regulation is a pivotal process in the control of neuronal functioning. In neurons resting free [Ca2+]j is in the range of KF-IO-8 M although the precise value is not well established (Kennedy and Thomas, 1996; Ross, 1993; Bassani et al, 1995). Despite this 2 low [Ca2+];, the total concentration of Ca2+ within a neuron is often in the millimolar range due to the large amounts of Ca2+ existing conjugated to Ca2+ binding proteins and non-specific Ca2+ binding sites (Widdowson and Dickerson, 1964). This observation stems from experiments by Hodgkin and Keynes (1957) who injected radioactive Ca2+ into squid giant axons and compared the rate of diffusion in the axoplasm versus that in solution. In complete contrast to the low levels of free [Ca2+];, the concentration of free extracellular Ca2+ ([Ca2+]0) is in the millimolar range, over four orders of magnitude (10,000 x) higher than [Ca2+];. This extremely high ratio of [Ca2+]G/[Ca2+]i, combined with the negative resting membrane potential, results in a large inward acting electrochemical gradient across the plasma membrane, the largest for any common inorganic ion (McBurney and Neering, 1987). Any changes in the permeability of the plasma membrane to Ca2+ will therefore produce significant fluctuations in [Ca2+]j which may lead to neuronal degeneration or neuronal death. A precise control of [Ca2+]; is therefore crucial in maintaining neuronal viability and functioning (Farber, 1981; Nelson and Foltz, 1983; Carafoli, 1987). II. Ca2+ entry into neurons There are two principal mechanisms by which [Ca2+]; is increased in neurons; Ca may enter via gated channels or Ca2+ may be released from intracellular stores (Carafoli, 1987; Simpson et al, 1995). Two principal types of gated Ca2+-channels exist; voltage gated channels, activated by membrane depolarization, and ligand gated channels, activated by the binding of extracellular ligands. 3 Voltage Gated Ca2^-Channels Voltage gated Ca2+-channels (VGCC) are the principal physiological entry point for Ca2+ during action potential conduction. The ability of VGCCs to translate action potentials into neurotransmitter release depends upon a variety of factors, such as the channels' conductance, proximity to release sites, density, response to depolarization and regulation by second messengers and transmitters (Regehr and Mintz, 1994). The four principal subtypes of VGCC can be distinguished based on their biophysical, pharmacological and structural properties (Bean, 1989; Hess, 1990; Miller, 1992; Tsien et al, 1988). The first attempts at categorizing voltage gated channels were made by Nowycky et al. (1985) and were based On observations of Ca2+-currents in chick dorsal root ganglion cells. Three channels were identified, a low threshold current which was named the T-type current, along with two higher threshold currents which were named the L and N-type currents. These channels were distinguishable not only on their structural properties but more importantly based on their blockade by pharmacological agents. T-type channels are sensitive to blockade by Ni2+, verapamil, phenytoin and amiloride whereas L-type channels can be modulated by dihydropyridines (DHPs) along with a number of cardiovascular-regulating agents (Bean, 1989; Nowycky et al, 1985; Miller, 1992). N-type channels are blocked by co-conotoxin GVIA (co-CgTx), a toxin derived from the venom of the piscivorous marine mollusk Conus geographus (Hirning et al, 1988; Plummer et al., 1989) and similar to the L-type channels, N-type channels are blocked by Cd2+ and Ni2+ (Tsien et al, 1988). A fourth type of channel was later identified and, based on the high numbers found in Purkinje cells (as compared with the low numbers found in other neuronal cells), was termed the P-type channel (Llinas et al, 1989; Regan et al, 1991; Regan, 1991). P-type channels are insensitive to DHP's and co-CgTx and are selectively blocked by co-Aga-IVA, isolated from the venom of the funnel-web spider (Mintz et al, 1992). Recently, with the advent of molecular 4 cloning, attempts have been made to classify VGCC based on their structure and sequence. For example Snutch et al. (1990) classified channels into four groups (A, B, C and D) based on their hybridization patterns to rat brain mRNAs. These four groups can be directly correlated with previous classification gropus, for example group A channels correspond with P-type channels while groups C and D correspond with L-type channels. Southern blot analysis and DNA sequencing suggest that each class of cDNA represents a distinct gene or gene family with classes A and B along with classes C and D being virtually homologous to each other. This suggested that all Ca2+-channels evolved from a single ancestral gene and the diversity in Ca2+-channels has arisen through gene duplication and subsequent divergence (Tsien et al, 1991). Ligand Gated Ca2+-Channels Ligand gated Ca2+-channels (LGCC) refer to channels which open in response to the binding of an extracellular ligand. The two subtypes of LGCC are those activated by the neurotransmitters glutamate or acetylcholine. Based on pharmacology, the glutamate receptor family can be further subdivided into two major subtypes; the NMDA and non-NMDA receptor activated channels. NMDA channels are opened by the agonist Af-methyl-D-aspartate while non-NMDA channels open in response to a-amino-3-hydroxy-5-methyl-4-isoxazole priopionate (AMPA) or kainate. Some ligand gated channels are also voltage sensitive, for example, the NMDA channel responds to membrane depolarizations by releasing a Mg2+ block which normally prevents Ca2+ entry. It is this voltage-dependent Ca2+-entry which gives the NMDA channel its distinct role in long-term potentiation (Mayer and Westbrook 1987; Tsien and Tsien, 1990). 5 Intracellular Ca2+ Stores To date the general consensus is that a rise in [Ca2+], originates from the opening of VGCC or LGCC. However recent evidence indicates that a major component of the total Ca2+ signal appears to originate from intracellular Ca2+-stores (Simpson et al., 1993). As a result, intracellular Ca2+-stores, in particular those associated with the endoplasmic reticulum, play a crucial role in Ca2+-signalin'g as they not only act as a Ca2+-source by initiating signals but may also act as a Ca2+-sink by sequestering Ca2+ following an increase in [Ca2+]j. These stores are influenced by a variety of compounds, for example Ca2+ is released in response to adenosine triphosphate (ATP), inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphate (ADP) ribose, caffeine, or low concentrations of ryanodine, while Ca2+-release is inhibited by thapsigargin, cyclopiazonic acid (CPA), 2,5,-Di-(t-butyl)-l,4-benzohydroquinone (DBHQ), and high concentrations of ryanodine (Berridge, 1997). These stores may also be involved in a variety of neuronal processes, such as excitability, transmitter release, synaptic plasticity, gene expression and apoptosis (Holliday etal., 1991; Simpson etal., 1995). As [Ca2+]i often reaches micromolar levels following intense stimulation, neurons must possess effective mechanisms to sequester and extrude these potentially dangerous increases in [Ca2+]i (Werth and Thayer, 1994). Mechanisms by which a neuron can regulate [Ca2+]i include the plasma membrane Na+/Ca2+ exchanger (PM-Na+/Ca2+), the plasma membrane Ca2+-adenosine triphosphatase (PM-ATPase), the endoplasmic reticulum Ca2+-adenosine triphosphatase (ER-ATPase), mitochondria, and Ca2+-binding proteins (see Nicholls, 1986; Carafoli, 1987; Miller, 1991; Baimbridge et al, 1992 for reviews). Since Ca2+ may be involved in multiple neuronal activities within different neuronal regions (soma, neurites, growth cones, and terminal boutons), there exist substantial spatial and temporal variations of [Ca2+]i amongst 6 these regions which are manifested in the form of a heterogeneous distribution of Ca2+-channels and Ca2+-homeostatic mechanisms within the neuron (Katz and Miledi 1969; Ross et al, 1986; Miller, 1991; Carafoli, 1987; Reuter and Porzig, 1995). As a result of these spatial and temporal variations, little is known about the modulation of Ca2+-homeostatic mechanisms. Despite this uncertainty, the general principles of Ca2+-regulation, such as the mechanisms involved and their kinetics, should be applicable to different parts of the neuron and in general to most neurons (Blaustein, 1988). III. Intracellular Ca2+-homeostasis Initially the Ca2+ which enters via VGCC or LGCC may be buffered by 'short-term' buffers such as Ca2+-binding proteins or intracellular Ca2+-stores but eventually the increase in Ca2+ must be extruded across the plasma membrane. Mechanisms designed to regulate [Ca2+]i can be categorized as being either short-term buffers (1 and 2 below) or long term extruders (3 below): 1. Pumps which sequester Ca2+ into organelles such as the mitochondria or endoplasmic reticulum (ER). 2 Proteins, or other buffers, which bind Ca2+ within the cytoplasm. 3. Pumps or exchangers which extrude Ca2+ from the cell. 1. Ca2+ATPase There are two principal Ca2+-ATPases found within neurons; those located on the plasma membrane which pump Ca2+ out of the cell (PM-ATPases) and those located on intracellular 7 compartments which pump Ca2+ into intracellular stores (for example the ER-ATPase). Both of these ATPases use the energy generated by the hydrolysis of ATP to pump Ca2+ 'uphill' against its electrochemical gradient. a) PM-ATPase The first clue suggesting the presence of a Ca -dependent ATPase in eukaryotic cells was seen over thirty-five years ago by Dunham and Glynn (1961) who noticed a relationship between ATP activity and the movement of alkali metal ions. Schatzmann (1966) later showed that erythrocyte ghosts loaded with Ca2+ and ATP had a much higher Ca2+ efflux rate compared with erythrocyte ghosts loaded only with Ca2+. Since this efflux occurred against a concentration gradient Schatzmann inferred the presence of a pump which used energy to overcome this gradient. The PM-ATPase has since been shown to be present on the plasma membrane of all eukaryotic cells and its mechanism has been shown to involve ATP-phosphorylation of an aspartic acid residue in a Ca2+-dependent manner followed by the translocation of one Ca2+ across the plasma membrane (Knauf etai, 1974; Katz and Blostein, 1975; Carafoli, 1991). The PM-ATPase is in fact a Ca2+/H+ exchanger, exchanging one extracellular FT" with one intracellular Ca2+ (Niggli et al, 1982; Smallwood et al, 1983; Milanick et al, 1990; Carafoli 1991; Schwiening et al, 1993). The PM-ATPase has an essential requirement for Mg2+ and is inhibited by vanadate (Kd ~ 2-3 uM), which blocks an aspartic acid phosphorylated intermediate, La3+, which increases the steady-state level of the phosphoenzyme and intracellular acidification and extracellular alkalinization, which influence the H+-gradient (Carafoli 1987 and 1991). Calmodulin, by way of a direct interaction with the ATPase, increases the activity of the PM-ATPase (Gopinath and Vincenzi, 1977) and increases the affinity for Ca2+ by up to 30 fold 8 and the Vmax by up to 10 fold (Lynch and Cheung, 1979; Carafoli, 1991). The PM-ATPase is also activated by a variety of factors such as phospholipids from the inositol phosphate pathway (phosphatidyl inositol and its mono and di-phosphate derivatives), along with long chain polyunsaturated fatty acids (Ronner et al, 1977). b) endoplasmic reticulum ATPase Many tissues possess ATP-dependent Ca2+ sequestration mechanisms. The first ATP-dependent intracellular Ca2+ pump to be discovered was the Ca2+-ATPase of the sarcoplasmic reticulum (SR) (Ebashi and Lippman, 1962). Using X-ray microanalysis it was later shown that a similar ATPase existed in the ER. Following the cloning of cDNA's encoding homologous ATPases, the presence of several isoforms of this Ca2+-pumping ATPase was discovered in both the SR and the ER (MacLennan et al, 1985; Brandl et al, 1986). With the exception of the SR in muscle, these stores have a small Ca2+-sequestration capacity and are insensitive to the mitochondrial inhibitor ruthenium red. These ER Ca2+-stores are influenced by a variety of factors; they are released by A23187 and high [Ca2+]j, while their uptake of Ca2+ is modulated by calmodulin, cAMP-dependent protein kinases, vanadate and ryanodine (Carafoli, 1987). A variety of specific inhibitors to the ER-ATPase exist, the most potent being thapsigargin (Tg). This tumor-promoting lactone was originally extracted from the resin of the umbelliferous plant Thapsia garganica and used initially for its ability to increase [Ca2+];. Later, it was determined to be a specific inhibitor of the ER-ATPase and not other cation ATPases such as the PM-Na+, K+ and Ca2+-ATPases (Rasmussen et al, 1978; Thastrup et al, 1990; Inesi and Sagara, 1994). Other inhibitors of the ER-ATPase include CPA, a toxic metabolite produced by the molds Pencillium cylopium and Aspergillus flavus and DBHQ (Holzapfel, 1968; Goeger et al, 1988; Moore et al, 1987). 9 2. PM-Na+/Ca2+ exchanger The PM-Na+/Ca2+ exchanger was first identified in heart muscle (Reuter and Seitz, 1968) and neuronal tissue (Blaustein and Hodgkin, 1969), and has since been found on the plasma membrane of all cell types, with the exception of erythrocytes. The PM-Na+/Ca2+ exchanger was originally postulated to be an electroneutral exchanger with 2 Na+ being imported for every 1 Ca2+ extruded, but Blaustein and Hodgkin (1969) demonstrated that the energy content of the transmembrane Na+ gradient was not large enough to maintain the large Ca2+-gradient across the plasma membrane. Following this revelation, experiments by Reeves and Sutko (1979) on sarcolemmal vesicles unequivocally demonstrated that the PM-Na+/Ca2+ exchanger exchanged 3 Na+ for every 1 Ca2+ extruded from the cytosol, producing one inward positive charge per cycle. The PM-Na+/Ca2+ exchanger is sensitive to changes in the Na+ or Ca2+-gradient or changes in the membrane potential and by changing either of these factors it is possible to induce the reverse mode of the exchanger, thereby promoting Ca2+-influx (Carafoli, 1987). ATP is needed indirectly as the Na+ which enters the neuron via the exchanger must eventually be extruded across the PM by the Na+/K+-ATPase. The Na+/Ca2+ exchanger has an absolute requirement for Na+, and Na+ substitutes such as Tris, choline, Af-methyl-D-glucamine, or Li+ are ineffective. There are no potent and specific inhibitors of the Na+/Ca2+ exchanger, although some compounds such as doxorubicin and its derivatives along with amiloride and its derivatives such as 3',4'-dichlorobenzamil (DCB) have been shown to have inhibitory actions on the exchanger (Caroni et al, 1981; Siegl et al, 1984). 3. Mitochondria 10 The principal role of mitochondria is to produce ATP, the common organic cellular fuel. Mitchell (1966) postulated the following series of reactions in order to explain ATP production. Products from the metabolism of sugars, carbohydrates, amino acids and lipids are used as carbon sources in the tricarboxylic acid (TCA) cycle. NADH and FADH2, products from the TCA cycle, serve as reducing equivalents in the electron transport chain (ETC) located on the inner mitochondrial membrane. At 3 points along the ETC, energy obtained from oxidation is used to pump H+ outwards across the inner mitochondrial membrane thereby creating an inward acting electrochemical gradient. When FF1" passes down this electrochemical gradient, through a pore in the ATP-synthase, ADP is phosphorylated forming ATP. Mitochondria take up calcium via a uniporter driven by the large electrochemical gradient across the inner membrane (Akerman et al., 1977; Carafoli, 1987; Gunter and Pfeiffer, 1990; Werth and Thayer, 1994). Calcium exits the mitochondria via a Na+/Ca2+ exchanger and Na+ is then removed via a Na+/H+ exchanger, the net result being that two H+ ions are prevented from entering the mitochondria via the ATP-synthase. As a result of mitochondrial Ca2+ sequestration, ATP production is decreased and the cycling of Ca2+ back and forth across the inner mitochondrial membrane will uncouple electron transport from ATP synthesis. Mitochondria are believed to sequester Ca2+ for two principal reasons. Firstly, they protect the neuron against the damaging effects of prolonged Ca2+ elevation, and secondly, they control metabolic processes such as the activation of Ca2+-sensitive dehydrogenases which influence ATP production (Gunter et al, 1994; Rizzuto et al, 1994; Hehl et al, 1996). The observation that mitochondria take up Ca2+ was made by De Luca and Engstrom (1961) and Vassington and Murphy (1962), and has since been demonstrated in a variety of neuronal 11 preparations (Sanchez and Blaustein, 1987; Stuenkel, 1994; Thayer and Miller, 1990; White and Reynolds, 1995). Early evidence, using isolated mitochondria, suggested that there existed a mitochondrial 'set-point' below which Ca2+-sequestration does not occur. However, above this 'set-point' in [Ca2+]i, traditional neuronal homeostatic mechanisms are saturated and mitochondria begin to sequester Ca2+. Mitochondria persist in sequestering Ca2+ until [Ca2+]; is reduced to a level below this 'set-point' at which time Ca2+ is released by the mitochondria to be extruded by the now unsaturated neuronal homeostatic mechanisms (Thayer and Miller, 1990). In experiments on isolated mitochondria, this set-point has been estimated to be in the range of 0.75 - 1.0 uM suggesting that mitochondria only sequester Ca2+ in rare pathological situations where [Ca2+]i is raised above physiologically 'normal' levels (Nicholls 1985; Carafoli 1987; Kostyuk et al, 1989; Thayer and Miller, 1990). However more recent evidence, obtained in whole cells, demonstrates that firstly, this mitochondrial 'set-point' may be lower than previously reported (Werth & Thayer, 1994), and secondly, that microdomains of high [Ca2+]; exist close to the mitochondria (Rizzuto et al, 1994) lending credence to the idea that mitochondria may take up Ca2+ following apparently minimal increases in [Ca2+]i (Gunter et al, 1994; Werth and Thayer, 1994; Kiedrowski and Costa, 1995; White and Reynolds, 1995; Hehl et al, 1996; Herrington et al, 1996; Park et al, 1996). The role of mitochondria in Ca2+-homeostasis can be. examined by the use of cell-permeant mitochondrial Ca2+-uptake inhibitors. Rotenone, an electron transport chain inhibitor, slowly decreases the mitochondrial electrochemical gradient, whereas the uncouplers carbonylcyanide /?-trifluoromethoxyphenylhydrazone (FCCP) and carbonylcyanide m-chlorophenylhydrazone (CCCP), both protonophores, permeabilize the inner mitochondrial membrane thereby rapidly eliminating the mitochondrial electrochemical gradient. 12 4. Ca2+ Binding Proteins The ability of Ca2+-binding proteins to buffer rises in intracellular Ca2+ depends on a number of factors. Firstly, the amount of protein present within the neuron, secondly, the Kd of the protein for Ca2+, thirdly, the forward rate constant Kon ('speed' of binding to Ca2+), and fourthly, the mobility or distribution of the protein within the cytoplasmic compartment (Sala and Hernandez-Cruz, 1990). The first Ca2+-binding protein to be studied in detail was parvalbumin, a muscle protein found at high concentrations in fish, amphibia, reptiles and mammals. Kretsinger and Nelson (1973) deduced the mechanism by which parvalbumin binds Ca2+ and in doing so established some general principles for the interaction of Ca2+ with Ca2+-binding proteins. From crystallized parvalbumin they deduced the existence of repeat domains which bound Ca2+ specifically and effectively; each domain contained a loop of 10-12 amino acid residues with two a-helixes aligned perpendicularly to each other, this helix/loop/helix peptide sequence, coined the 'EF-hand', has been found in over twenty Ca2+-binding proteins and is crucial for the high affinity binding of Ca2+to Ca2+-binding proteins Based on function, Ca2+-binding proteins can be divided into two groups. Those which modulate the activity of enzymes and other cellular functions are known as 'trigger' proteins, while those whose primary function is thought to be the buffering of increases in [Ca2+]; are known as 'buffer' proteins (Baimbridge et al, 1992). An example of a 'trigger' protein is calmodulin, present in all eukaryotic cells. When complexed with Ca2+, calmodulin interacts with a variety of cellular enzymes such as adenylate cyclase, cyclic nucleotide phosphodiesterase, phosphorylase b kinase, phospholamban, and of particular importance to Ca2+-homeostasis, the PM-ATPase. An example of the 'buffer' variety of Ca2+-binding proteins is calbindin-D28K (CaBP), first identified in the chick gut but later found to exist predominantly 13 in the nervous system of mammals (Wasserman and Taylor, 1966; Baimbridge et al, 1992). Lledo et al. (1992) found that GH3 cells transfected with CaBP exhibited lower Ca2+-entry through VGCC and were better able to reduce Ca2+-transients evoked by voltage depolarizations as compared with 'CaBP -free' control cells. In addition, cells containing CaBP showed no changes in baseline Ca2+-levels as compared with CaBP free cells, suggesting that CaBP is only important during situations in which [Ca2+]i is above resting levels. Despite this lack of an effect on baseline Ca2+-levels, CaBP, as a result of its high affinity for Ca2+ (Ka ~ 500 nM) and high intracellular levels (up to 0.1 - 0.2 mM in some neuronal populations), could affect many aspects of the kinetics of Ca2+-transients. For example the peak [Ca2+], achieved, the rate of rise and decay of [Ca2+]i, neuronal excitability, action potential duration, neuronal 'bursting' activity (by inhibiting the Ca2+-dependent inactivation of voltage gated Ca2+-channels) and neuronal protection against the harmful effects of high [Ca2+]; may all be influenced by CaBP (Oberholtzer et al, 1988; Baimbridge et al, 1992; Chard et al, 1993) IV. Overview of Ca2+-homeostatic methods Although the presence of the above mentioned Ca2+-homeostatic mechanisms has been well documented, the relative contribution of each of these processes to overall Ca2+-homeostasis is not well understood. Depending on the cell type studied, different groups have found diverse roles for individual Ca2+-homeostatic mechanisms. Initial work by Dipolo and Beauge (1979) suggested that the PM-Na+/Ca2+ exchanger and the PM-ATPase acted in tandem to reduce increases in [Ca2+]j. They characterized the PM-ATPase as a high affinity/low capacity pump and the PM-Na+/Ca2+ exchanger as a low affinity/high capacity exchanger. As a result, the PM-Na+/Ca2+ exchanger was postulated to be responsible for the initial fast phase of the removal of large Ca2+-loads while the PM-ATPase was postulated to be responsible for the secondary 14 restoration of [Ca2+]j to baseline levels (Dipolo and Beauge, 1979; Sanchez-Armass and Blaustein, 1987; Ahmed and Connor, 1988; Blaustein, 1988; Thayer and Miller, 1990; Benham et al, 1992; Gleason et al, 1995; Werth et al, 1996). Others have shown that the Na7Ca2+ exchanger plays a secondary role in reducing [Ca2+]; levels while other mechanisms, such as mitochondria or the PM-ATPase, form the principal Ca2+-extrusion mechanisms (Bleakman et al, 1993; Kiedrowski and Costa, 1995; Mironov, 1995; Herrington et al, 1996). Mitochondria, initially thought to buffer [Ca2+]; only during pathological conditions of [Ca2+]i overload (Kostyuk et al, 1989; Thayer and Miller, 1990), have recently been implicated as an important mechanism for the removal of minimal increases in [Ca2+]i (Rizzuto et al, 1992; Gunter et al, 1994; Werth and Thayer, 1994; Kiedrowski and Costa, 1995; White and Reynolds, 1995; Hehl et al, 1996; Herrington et al, 1996; Park et al, 1996). As a result, it is clear that Ca2+-homeostasis is a complicated and dynamic process involving a large number of mechanisms, each of which may be activated at different times by differing factors. V. Experimental methods for increasing [Ca2+Ji In order to study the Ca2+-homeostatic mechanisms present within neurons, it is necessary to induce an increase in [Ca2+]i, which is in turn controlled by an interplay between the influx and efflux of Ca2+. To date, when imaging changes in [Ca2+]j from multiple neurons simultaneously, two principal methods have been used to increase [Ca2+];. Both of these methods require the supervision of solutions containing either high K+, which depolarizes the membrane and activates VGCC, or specific ligands whose receptors are coupled to Ca2+-permeable channels (LGCC), such as the iV-methyl-D-aspartate (NMDA) channel. In either case, the responses are relatively slow and during the period when Ca2+ is entering via VGCC or 15 LGCC, Ca2+ -homeostatic mechanisms are already initiated in an attempt to reduce the increase in [Ca2+];. The concurrence of these two opposing effects therefore makes Ca2+-decay kinetics very difficult to interpret. Additionally, it has been suggested that the mechanism(s) of Ca2+-homeostasis may be dependent upon the method used to increase [Ca2+]j. For example, White and Reynolds (1995) demonstrated that, depending on whether glutamate or high K+ was used to increase [Ca2+]b neurons utlized different Ca2+-homeostatic mechanisms. Ideally it would be best to examine Ca2+-homeostasis following a very rapid increase to peak [Ca2+]; and this has been made possible with the development of caged Ca2+-compounds. Following a brief exposure to UV light, [Ca2+];can be raised above physiological levels virtually instantaneously, thereby negating the slow rise to peak Ca2+-levels. These caged compounds are particularly useful because the flash of light can be easily controlled with respect to timing, 2+ location, duration, and amplitude (Adams and Tsien, 1993). Numerous properties of caged Ca -compounds must be taken into consideration when they are used in an intracellular environment. These include their pH sensitivity, their affinity for Ca2+ and selectivity of Ca2+-binding with respect to other ions, especially Mg2+, the relative binding of the native molecules with respect to their photolysis products and the quantum yield for Ca2+-release (i.e. the proportion of caged Ca2+-compound photolysed by UV light). Currently the two most popular caged Ca2+-compounds are DM-nitrophen and nitr-5 (Nerbonne, 1996). A relatively new caged Ca2+-compound, nitrophenyl-EGTA (NP-EGTA), meets a number of important criteria not previously attained by molecules such as nitr-5 or DM-nitrophen. NP-EGTA has been used successfully to induce the endocytosis of secretory granules in mouse pancreatic P-cells (Eliasson et al, 1996), contract chemically skinned skeletal muscle 16 fibres in rabbit (Ellis-Davies and Kaplan, 1994) and induce exocytosis in rat pituitary melanotrophs (Parsons et al, 1996). Due to the low affinity for Mg2+, the low affinity of the photolysis products for Ca2+ and the large percentage of NP-EGTA broken down during photolysis, NP-EGTA was the compound of choice for inducing increases in [Ca2+]; for the purposes of this study. VI. Effects of pH on Ca2+homeostatic mechanisms. Neurons possess a variety of mechanisms designed to regulate intracellular pH (pH;). These mechanisms can be subdivided into two categories based upon their abilities to acidify or alkalinize the cytoplasm; acid extruders increase pH; while acid loaders decrease pH;. The predominant acid extruders are the NaV FT" exchanger (NHE), which exchanges one extracellular Na+ ion for one intracellular H+, and the Na+-dependent C17HC03" exchanger which exchanges one extracellular Na+ and one extracellular HCO3" for one intracellular CI" ion. The principal acid loaders are the Na+-independent C17HC03" exchanger, which exchanges one extracellular CI" with one intracellular HCO3" and the Na+/HC03_ co-transporters, which transports one extracellular Na+ ion and one extracellular HCO3" ion out of the cell. Different cell types rely on different pH regulatory mechanisms in order to maintain a constant pH;. For example, in rat sympathetic neurons, rat brain synaptosomes, cultured rat cerebellar Purkinje cells and rat neocortical neurons, the NHE appears to be the dominant acid extrusion mechanism, whereas in CA1 hippocampal pyramidal neurons, the NHE along with other mechanisms such as the Na+-dependent HCO37CI exchanger appears to be the dominant acid extrusion mechanisms (Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996). 17 Intracellular pH in the majority of cell types is kept constant within the range of 6.8 to 7.2 (Putnam, 1995). Any prolonged deviations from this range are known to result in cellular toxicity and/or cellular death (Kraig et al, 1987). In neurons, changes in prl have been shown to affect the activity of many enzymes or ion channels, the activities of VGCC or LGCC and the excitability of neurons (Chesler and Kaila, 1992). Additionally, cytoskeletal properties (cell shape and motility), cell-cell coupling, and membrane conductance are all dependent upon pH; (Putnam, 1995). Of particular importance to the present study is the influences of pH; on Ca -regulatory mechanisms within neurons. For example, lowering pH; inhibits the PM-ATPase (Carafoli, 1987), decreases the affinity of Ca2+-binding proteins for Ca2+ (Ingersoll and Wasserman, 1971), and decreases mitochondrial Ca2+-uptake (Gambassi et al, 1993). Additionally the K<j for Ca2+ of fluorescent Ca2+-indicator dyes along with caged Ca2+-compounds increases significantly (i.e. becomes less sensitive) as acidity increases (Martinez-Zaguilan et al., 1996) Researcher-induced changes in pH; have many times been overlooked and may produce uninterpretable and confusing results. For example, due to the lack of a specific blocker for the PM-Na+/Ca2+ exchanger, many groups have simply removed extracellular Na+ (Na+0) (Na+ substituted by choline, A^methyl-D-glucamine, or Li+) and in doing so have assumed that only the PM-Na+/Ca2+ exchanger is blocked (Benham et al, 1992; Sanchez-Armass and Blaustein, 1987). However, the NHE is also blocked leading to a decrease in pHj. Therefore, in any neuron in which the NHE is a major component of pH; regulation, pH; will be influenced by Na+0 substitution and the interpretation of Ca2+ recovery kinetics may be incorrect. This was demonstrated by Koch and Barish (1994) who found that acidification of the cytoplasm, due to blockade of the Na+/H+ exchanger using Na+ free media, resulted in a two-fold increase in the 18 time required for [Ca2+]i to return to baseline levels following glutamate activation. As a result, it is crucial for pH; to be maintained in order to ensure that the intracellular environment is kept constant and undisturbed. VII. Goals of this study This study had three purposes, firstly, to develop a reproducible method for loading both NP-EGTA and the fluorescent indicator dye fluo-3 into neurons, secondly, to develop a photolysis protocol which allowed for significant increases in [Ca2+]i following flash photolysis of NP-EGTA and thirdly, to investigate which Ca2+-homeostatic mechanisms are responsible for reducing [Ca2+]; back to baseline values following flash photolysis of NP-EGTA with particular emphasis of the relative roles of the PM-Na+/Ca2+ exchanger and the mitochondria. 19 METHODS AND MATERIALS I. Tissue culture All experiments were performed on primary cultures of 4-day postnatal hippocampal neurons derived from Wistar rats using the method of Huettner and Baughman (1986) as modified and described previously (Abdel-Hamid and Baimbridge, 1997). Cultures were used after 7-10 days in vitro (DIV). Glial multiplication was inhibited by a single addition of cytosine arabinoside (10 uM) to the culture medium 48 hours after plating. In specifying culture age, the plating day was counted as day zero in vitro (0 DIV). The growth medium was Eagle's minimum essential medium with N2 supplement (insulin 5 u.g ml"1, transferrin 100 ug ml"1, progesterone 20 nM, putrescine 100 uM, sodium selenite 30 nM). Media were obtained from Gibco (Grand Island, NY) and were replenished by replacing half the volume with fresh N2-supplemented medium twice weekly. II. Ca2*-imaging with fluo-3 and the use of NP-EGTA i. Loading Fluo-3 and NP-EGTA were both purchased as their acetoxymethyl (AM) esters from Molecular Probes Inc. (Eugene, Oregon). Fluo-3-AM was dissolved in dimethlysulfoxide (DMSO) to produce a 1 mM stock solution which was stored in 25 ul aliquots at -80 °C until use. NP-EGTA-AM was purchased in individually packaged 50 [ig aliquots and similarly stored. To prepare the loading solution, a 25 ul aliquot of fluo-3-AM and an additional 25 ul of DMSO were added to a vial of NP-EGTA-AM and vortex mixed. Using a fine pipette tip, 25 ul of this 20 mixture was added, while mixing vigorously with a vortex stirrer, to 4 ml of a balanced salt solution (BSS; NaCl 139 mM, KC1 3.5 mM, Na2HP04 3 mM, NaHC03 2 mM, HEPES acid 6.7 mM, HEPES-Na+ 3.3 mM, D-Glucose 11 mM, CaCl2 1.8 mM, Glycine 2 uM, tetrodotoxin 1 uM; pH 7.35) containing 0.05% bovine serum albumin (Sigma). The final concentrations of fluo-3-AM and NP-EGTA-AM were 3.1 uM and 8 uM respectively. In one experiment, nitr-5-AM (Chemica Alta LTD, Edmonton, Alberta, Canada; final loading concentration 12.5 uM), was substituted for NP-EGTA-AM. Individual coverslips, plated with neurons, were placed in a well of a 6-well culture dish and incubated for 1 hour at room temperature in the dark in 2 ml of this loading solution. They were then transferred to fresh BSS for at least 15 minutes prior to, or after mounting on a superfusion chamber. After loading, and for the duration of the experiment, the cells were maintained at 20 - 22 °C This loading protocol was established empirically to yield sufficient intracellular concentrations of fluo-3 for adequate imaging and sufficient NP-EGTA to yield a 2-4 fold increase in background corrected fluo-3 fluorescence following a 0.4 s exposure to UV light. Whenever possible, control and experimental data were collected from paired sister cultures loaded with identical solutions. ii. Imaging Fluo-3 fluorescence was measured using a Zeiss-Attofluor™ digital fluorescence imaging system controlled by Attofluor imaging software. The microscope used was a Zeiss Axiovert-10 fluorescent microscope which was equipped with a long distance 40x objective (Zeiss LD UV-Achroplan 0.6, Ph2) and a 100 W mercury arc lamp as a light source. The mercury lamp was used at full power for all of the experiments and no neutral density filters were in place. Excitation light for the fluo-3 was filtered through a 10 nm band pass 488 nm excitation filter 21 whose position was determined by a computer-controlled solenoid filter changer. The filtered light passed through a long band-pass dichroic mirror (FT-495) before being focused through a 40 x Neofluar objective (numerical aperture 0.75) onto the cells in the superfusion chamber. The emitted light was passed through a dichroic beam splitter before being filtered by a 510 nm long-pass filter. The imaging software operated on a dual-monitor IBM-compatible 80486-33 MHz which controlled the filter changer, camera gain, image processor, data processor, data display and data storage. One of the monitors displayed a pseudocoloured image of the field of view while the second monitor gave the raw emission intensity plotted against time. The photosensitive detector was a high sensitivity, charge-coupled device (CCD) camera and images were digitized to 8 bit resolution with a 512 x 480 pixel frame size on a Matrox-AT image processor. The coverslips containing the plated hippocampal neurons were mounted face-up in a superfusion chamber made in our departmental workshop. The inflow channel was connected to a perfusion pump while the outflow channel was connected to a suction line which removed all of the superfusate present above a level of 3 mm. Neurons were superfused at a rate of 2.4 ml min"1 at 20 - 22°C with Mg2+-free BSS. In order to select an appropriate field of view, a 12 V 100W halogen lamp was used to visualize the cells under phase illumination. Regions of interest (ROIs) were set at 10 x 10 pixels and placed over the soma of up to 99 neurons in field of view. The camera gain was set at a level in order to minimize camera saturation while at the same time maximizing image intensity. At the normal resting [Ca2+]j of our neurons, the baseline fluo-3 fluorescence was often very low, thus making the identification of neurons and the placement of ROIs difficult. In such cases, 2 ml of a 40 u,M NMDA solution in BSS was added through a pipette placed over the inlet 22 of the chamber. A maximum increase in fluo-3 fluorescence was seen approximately 10 to 15 seconds later, at which time an image was stored and the flow rate was then increased for 10 s to maximum (about 10 ml min"1) in order to wash out the NMDA. This stored image was then used for the placement of ROIs. In order to uncage NP-EGTA, the acquisition of fluo-3 fluorescence data was interrupted and the cells exposed to unfiltered UV light for 0.4 s by the placement of an empty filter holder in the excitation light path. Fluo-3 fluorescence data acquisition then continued ~ 0.6 s after uncaging. To minimize photobleaching, for the first 30 s after uncaging the acquisition rate was one image per second, for the next two minutes it was one image every two seconds, and for the final thirty seconds it was one image every three seconds. This total time of three minutes was, in most experiments, more than enough for the fluo-3 fluorescence to decay back to baseline values. Data was recorded from up to three well-separated fields on a single coverslip, ensuring that neurons were only exposed to a singular 0.4 s flash of UV light. iii. NP-EGTA data analysis Using a stand-alone DOS-based program ATTOGRAF (Atto Instruments Inc., version 5.41), regions of interest deemed acceptable were chosen for analysis. Neurons were deemed acceptable if the background corrected fluo-3 fluorescence, firstly, increased by 50 % or greater in response to NMDA (thereby excluding glial cells), secondly, increased at least two-fold in response to flash photolysis of NP-EGTA and, thirdly, returned within three minutes to 110 % of that recorded prior to flash photolysis (with the exception of those neurons treated with CCCP; see results below). For each neuron, the fluorescence intensity was normalized between a value 23 of 0 and 1 with 0 being the baseline value to which fluo-3 fluorescence decayed, and 1 being the fluorescence at the first time-point immediately following photolysis. The data was processed for a best fit exponential decay curve using Table Curve-2D™ which, in the majority of situations, was a double-exponential decay curve with: Rate=Ai expC"^l) + A2 exp(-t/x2) + C Where: t - time (s) Ai - amplitude of the first (fast) exponential il - time constant of the first (fast) exponential A2 - amplitude of the second (slow) exponential T2 - time constant of the second (slow) exponential C - constant Statistical analysis was carried out using Student's two-tailed t test with a 95 % confidence limit. In all cases unpaired t values were calculated with supplemental paired data added when appropriate. III. Ca2*-imaging using fura-2 i. Loading Fura-2-AM was obtained from Molecular Probes Inc. (Eugene, Oregon) and stored in 30 ug aliquots. 25 ul of anhydrous DMSO was added to a vial of fura-2-AM which was then added slowly, while vortex mixing, to 4 ml of BSS which had been supplemented with 0.05% bovine serum albumin. The mixture was then vigorously vortex mixed in order to disperse the fura-2-24 AM solution. Coverslips were then incubated for 1 hour at room temperature in 2 ml of this loading media. Neurons were transferred to fresh BSS for at least 15 minutes prior to, or after mounting on a superfusion chamber. 77. fura-2 data analysis Fura-2 measurements were made using a dual excitation ratiometric method on the same imaging system. Excitation light was fdtered through either 334 nm or 380 nm (10 nm band pass) excitation filters. Briefly the dye was excited alternately at either 334 or 380 nm (10 nm band pass filters) and the resulting fluorescence above 510 nm was recorded. The frequency of image acquisition was varied between 1 image every second to 1 image every 15 seconds depending on the experiment. Less frequent acquisition were used whenever possible to eliminate photo-bleaching of the fura-2. The ratios of background corrected emission intensities (/334//380) were converted to [Ca2+]; using the method of Grynkievicz et al. (1985) which involved the use of the following equation: [Ca2+];=PK4R-R^l [ Rmax " R] Where: P - the ratio of the measured fluorescence intensity of Ca free to Ca bound indicator at 380 nm. Ka - the dissociation constant of Fura-2 for Ca2+. R - the background corrected emission intensity ratio (/334^380)-Rmin - the limiting value of R when all the indicator is in the Ca2+-free form. Rmax - the limiting value of R when all of the indicator is in the Ca2+-bound form. 25 For our calibration experiment, the values of Rmin, Rmax, and (3 were 0.3, 8.6 and 11.9 respectively. These values were obtained by exposing neurons to 0 Ca2+ solutions (0 Ca2+: NaCl 139 mM, KC1 3.5 mM, Na2HP04 3 mM, NaHC03 2 mM, HEPES acid 6.7 mM, HEPES-Na+ 3.3 mM, D-Glucose 11 mM, Glycine 2 uM, tetrodotoxin 1 uM; EGTA 1 mM; pH 7.35) for 10 mins followed by the addition of 2 x 2 ml of this 0 Ca2+ solution which contained 10 |aM of the Ca2+-ionophore Br-A23187. Data was then collected for 30 min at an acquisition rate of 1 image/min at which time another 2 x 2 ml of this 0 Ca2+ solution with 10 uM Br-A23187 was added and data was collected for a further 15 min. The background subtracted ratio obtained following this 45 minute data acquisition period was considered Rmi„. Following this, 2 x 2 ml of BSS was added and the ratio obtained following 5 min incubation in this media was considered Rmax-IV. Ca2+-imaging using fura-red i. Loading Fura-Red-AM was obtained from Molecular Probes Inc. (Eugene, Oregon) and dissolved in DMSO to give a 0.45 mM stock solution which was stored in 75 uM aliquots at -60 °C. 75 ul of this Fura-Red-AM solution was added to 4 mL of BSS to produce a final loading concentration of 8 pM Fura-Red-AM. This solution was used to load neurons on a single coverslip for 1 hour at room temperature. They were then transferred to fresh BSS for at least 15 minutes prior to, or after mounting on a superfusion chamber. 26 ii. fura-red data analysis Fura-Red measurements were made using a dual excitation ratiometric method available on the same imaging system. Briefly, the dye was excited alternately at 452 nm and 488 nm and the resulting fluorescence above 510 nm was recorded. The frequency of data acquisition was kept to a minimum in order to avoid photo-bleaching the fura-red. The ratio of background corrected emission intensities (^452^488) was then converted to [Ca2+]i values with the following equation: [Ca ]i = [3 KH f R - Rmin 1 [ Rmax - R] Where: P - the ratio of the measured fluorescence intensity of Ca2+-free to Ca2+-bound indicator at 488 nm. 2_j_ Kd - the dissociation constant of Fura-Red for Ca . R - the background corrected emission intensity ratio (£452/^488)-Rmin - the limiting value of R when all the indicator is in the Ca2+-free form. Rmax - the limiting value of R when all of the indicator is in the Ca2+-bound form. Using the same calibration method as for fura-2 (above), values of Rmin, Rmax and P were 1.28, 6 and 2.85 respectively. 27 V. pH imaging using BCECF i. Loading 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-AM was obtained from Molecular Probes Inc. (Eugene, Oregon) and dissolved in DMSO to give a 1 mM stock solution which was stored in 60 u.1 aliquots at -60 °C. 5 ul of the BCECF-AM solution was taken from the stock solution and added, while vortex stirring, to 2.5 ml of BSS to produce a final concentration of 2 uM BCECF. This loading solution was transferred to one well of a six well tissue culture plate and used to load neurons on a single coverslip. Loading was for 30 minutes at room temperature (Baxter and Church, 1996). Neurons were then transferred to fresh BSS for at least 15 minutes prior to, or after mounting on a superfusion chamber. ii. BCECF data analysis BCECF measurements were made using a dual excitation ratiometric method available on the same imaging system. The advantages of a ratiometric method for estimating pH; are that the ratio obtained is insensitive to changes in optical path length, local probe concentrations, illumination intensity and photobleaching (Bright et al, 1987). Full details of the methodology can be found in Baxter and Church (1996). Briefly, the dye was excited alternately at 488 nm and 452 nm and the resulting fluorescence above 510 nm was recorded. The intensity of light illuminating the neurons was reduced by decreasing the power output of the mercury arc lamp to half that used for the fluo-3 experiments and by the addition of neutral density filters to the excitation light path. The frequency of image acquisition was kept constant at 1 image every 10 28 seconds and data was recorded until steady baseline readings were observed. Analysis was restricted to those neurons able to retain the fluorescent indicator (as judged by raw emission intensity values) throughout the entire course of the experiment (see Schwiening and Boron, 1994). In order to correct for minor differences in dye behavior between our in vitro calibration solution and the cytosol, BCECF ratios measured from each cell were normalized to an intracellular ratio recorded at the end of the experiment after a 15 minute exposure of the cell to a pH 7.0 medium containing 10 uM of the FfMonophore nigericin (Chaillet and Boron, 1985). This resulted in a normalization of the ratios with a value of 1.0 being equivalent to a pH; of 7.0. The normalized ratios were then transformed into pH; values using the equation: pHi= log[(Rn-Rn(min))/(Rn(max)-Rn)] + pKa Where: Rn is the normalized ratio R-n(min) ls the normalized minimum ratio obtained from the standard curve. R-n(max) 's tne normalized maximum ratio obtained from the standard curve. pKa is the acid dissociation constant. In a total of nine calibration experiments, the mean values of Rn(min)> R-n(max)> anc* pKa were 0.47 ± 0.07, 2.06 ± 0.20, and 7.30 ± 0.07 respectively. These values were obtained by exposing neurons to a variety of HEPES-buffered solutions of different pH's (ranging from 5.5 to 8.5) at room temperature. These solutions all contained 10 uM nigericin, a charged electron carrier ionophore which equilibrates pH; with pH0 as long as the intracellular and extracellular K+ activities are equal and high (Chaillet and Boron, 1985). 29 RESULTS I. Loading conditions for fluo-3 and NP-EGTA The first objective of this study was to develop a method which used NP-EGTA to induce increases in [Ca2+]j with the fluorescent indicator fluo-3 being used to detect and record Ca2+-levels. The fluorescent indicator fluo-3 was the indicator dye of choice as a result of complications which arise when more traditional ratiometric dyes are used in conjunction with NP-EGTA (see Discussion). In order to develop correct loading parameters for both fluo-3 and NP-EGTA, many empirical trials were performed and the following parameters were varied: Table 1: Loading parameters investigated for fluo-3-AM and NP-EGTA-AM PARAMETER CONDITIONS BEST CONDITION 1. Loading time 30, 45, 60, 80 and 120 mins 60 mins 2. [Fluo-3-AM] ~ 1, 3, 5, 10 uM 3 uM 3. [NP-EGTA-AM] ~ 1, 4, 6, 8, 10, 14 uM 8 uM 4. Loading temperature 23 °C and 37 °C 23 °C 5. Photolysis Wavelength 334 ± 5 nm, 360 ± 5 nm and Unfiltered UV light Unfiltered UV light 6. Photolysis Time 0.1, 0.15, 0.2, 0.25, 0.35, 0.4 and 0.5 s 0.4 s 7. Pluronic F-127 Absent or 0.02 % Absent 30 II. Neuronal Ca2+ responses to NMDA and caged Ca2+ compound In all experiments, neurons were identified on the basis of their Ca2+ response to a transient (5 s) exposure to 40 uM NMDA (Fig. 1). This produced a peak in fluo-3 fluorescence 5-15 s after the onset of exposure and the recovery to baseline from the peak value was relatively slow and in the order of 250-400 s. For comparative purposes, responses to NMDA recorded with the ratiometric dye fura-2 yielded peak [Ca2+]; in the range of 600 nM and resting [Ca2+]i of ~ 80 nM in our neurons (see Fig. 5; Abdel-Hamid and Baimbridge-submitted, Abdel-Hamid and Baimbridge-in press). In contrast, a flash photolysis of NP-EGTA produced a rapid peak fluo-3 fluorescence which was already recovering at the time of the first measurement following photolysis (~ 0.6 s). Thereafter, the recovery was rapid and usually complete within 3 minutes. Since unphotolysed and unbound NP-EGTA could contribute to Ca2+-buffering following photolysis, we examined the effect of successive 0.4 s flashes. The maximum number of uncages on a single field was two; any subsequent uncages produced negligible increases in fluo-3 fluorescence over baseline values. A second uncage on the same field of neurons resulted in a smaller peak indicating that approximately 60% of the loaded NP-EGTA was photolysed by the first uncage (Fig. 2). When a similar experiment was repeated with continuous supervision of a medium in which Na+ was replaced by NMDG immediately after the first of two uncagings (Fig. 3), the second response was less than 25 % of the first, indicating that 0 Na+ conditions, which reduce pH, (see Fig. 9), also reduce the amount of releasable Ca2+ from NP-EGTA. This is consistent with the known sensitivity of EGTA (and its derivative NP-EGTA) to pH, especially below a value of pH 7.0 (Kao, 1994). Based upon the reported Kd values of NP-EGTA and nitr-5 (~ 80 nM and ~ 145 nM respectively) we would anticipate that approximately 50 % of the NP-EGTA molecules would be in a Ca2+ bound state at the resting [Ca2+]j of our neurons (-80 nM, see Fig. 5 and 6) 31 Figure 1: Typical control response in a field of neurons to NMDA application and flash photolysis. Mean response of a field of neurons (n=12) loaded with both fluo-3 and NP-EGTA exposed for 5 s to 40 uM NMDA followed by a 0.4 s flash of unfdtered UV light (Uncage). Resting Ca2+-levels increased following NMDA application. In this, and all subsequent similar figures, an increase in [Ca2+]i is indicated by an increase in background corrected fluo-3 fluorescence intensity. Following the removal of NMDA, Ca2+-levels decayed back to baseline values over the course of the next 500s. When a 0.4 s flash of UV light was used to uncage the NP-EGTA, the resulting peak Ca2+ level decayed back to baseline values at a much quicker rate compared to the response following NMDA, with baseline Ca2+-values being restored to within 300 s. A Time (s) 33 Figure 2: Typical response in a field of neurons to NMDA followed by two consecutive uncages. Mean response of a field of neurons (n=10) loaded with both fluo-3 and NP-EGTA exposed for 5 s to 40 uM NMDA followed by two consecutive 0.4 s flashes of unfiltered UV light (U). Ca2+-levels increased following NMDA application and then returned to near baseline values. The two responses to uncaging of NP-EGTA decayed to near baseline values within 300 s. The markedly reduced peak reached as a result of the second uncaging demonstrates that the majority of the NP-EGTA was photolyzed during the initial 0.4 s flash. Time (s) 35 Figure 3: The influence of pH on NP-EGTA Mean response of a field of neurons (n=14) loaded with both fluo-3 and NP-EGTA exposed for 5 s to 40 pM NMDA followed by two 0.4 s uncages (U). Following the first uncage, all external Na+ was replaced with NMDG, as a result, the second uncaging was performed during a decrease in pH; (see Fig. 9). The second photolysis peak is much smaller than is normally expected for a second uncage (see Fig. 2 for comparison) demonstrating the pH sensitivity of NP-EGTA binding with Ca2+. 36 125 n NMDA U CO c (D £ c 00 oo XT 0Na+-NMDG 0 200 400 600 800 1000 1200 1400 Time (s) 37 compared with < 25 % in the case of nitr-5 (Nerbonne, 1996). This is consistent with the relatively modest increase in fluo-3 fluorescence in response to photolysis of nitr-5 unless the uncaging was timed with the recovery phase of a substantial Ca2+ response to NMDA (Fig. 4). These results emphasize the advantage of using NP-EGTA rather than nitr-5. Although the fluorescent indicator dye fluo-3 may be calibrated, the method is inaccurate and not widely accepted, and was not attempted in our experiments (Minta et al, 1989; Kao et al, 1989; Kao, 1994). In order to obtain an indication of the [Ca2+]i reached during photolysis, the ratiometric and calibratable dye fura-2 was used. In 23 neurons loaded with fura-2 and NP-EGTA, resting [Ca2+]i was ~ 90 nM (Fig. 5). Following 40 uM NMDA application, [Ca2+]; reached 950 nM before decaying back to resting levels of - 90 nM. Uncaging using a 0.4 s flash of unfiltered UV light, produced a peak [Ca2+]i of 530 nM which subsequently decayed back to baseline levels of ~ 100 nM. In a second experiment increasing the photolysis time to 0.5 s (Fig. 6), resulted in a peak [Ca2+]i of 720 nM (n=12) before decaying back to resting [Ca2+]; of - 100 nM,with NMDA application producing a peak of 1050 nM. As a result of the complications associated with simultaneous using fura-2 and NP-EGTA (see discussion), we attempted to use the ratiometric dye Fura-Red to determine the peak [Ca2+]; reached following flash photolysis. Baseline [Ca2+]i was found to be - 100 nM (Fig. 7), consistent with those found using fura-2, while peak [Ca2+]i immediately following flash photolysis was seen to be - 800 nM. Both of these ratiometric dyes indicated that the peak [Ca2+]i observed immediately following photolysis was similar to that found during a 5 s application of 40 uM NMDA, indicating that the loading parameters used in this study did not produce an excessive increase in [Ca2+];. Although the [Ca2+]; found immediately following flash photolysis was not excessive, it does not reflect the true peak [Ca2+]; reached following flash photolysis as the first data point was obtained - 0.6 s 38 Figure 4: The effects of the caged Ca -compound nitr-5. Records from three individual neurons loaded with both nitr-5 and fluo-3 exposed for 5 s to 40 uM NMDA followed by a 0.4 s flash of UV light (U). Photolysis of nitr-5 produced negligible increases in Ca2+-levels unless the [Ca2+]; was elevated during the photolysis demonstrating the reduced affinity of nitr-5 for Ca2+. 39 Time (s) 40 Figure 5: Response seen to 0.4 s flash in fura-2 and NP-EGTA loaded neurons. Mean response of a field of neurons (n=14) loaded with both fura-2 and NP-EGTA exposed for 5 s to 40 uM NMDA followed by a 0.4 s flash of UV light (U). Baseline [Ca2+]; was ~ 80 nM, following NMDA application [Ca2+]i reached ~ 900 nM. Following flash photolysis of NP-EGTA [Ca2+]i reached ~ 500 nM. + CM 03 O 1200 1000 800 600 400 -200 -0 NMDA Time (min) 42 Figure 6: Response seen to 0.5 s flash in fura-2 and NP-EGTA loaded neurons. Mean response of a field of neurons (n=10) loaded with both fura-2 and NP-EGTA exposed for 5 s to 40 uM NMDA followed by a 0.5 s flash of UV light (U). Baseline [Ca2+]; was ~ 80 nM, following NMDA application [Ca2+]; reached ~ 900 nM. Following flash photolysis of NP-EGTA [Ca2+]i reached ~ 700 nM. This value of - 700 nM was similar to that obtained with the use of Fura-Red and was larger than the peak [Ca2+]; reached following a 0.4 s flash (see Figure 5). Reasons for the decreased amount of Ca2+ released during the 0.4 s flash are outlined in the discussion. + CM 03 o 1200 1000 i 800 600 400 200 NMDA 0 0 1 2 3 4 Time (min) 44 Figure 7: Response seen to a field of neurons loaded with fura-red and NP-EGTA. Mean response of a field of neurons (n=16) loaded with both fura-red and NP-EGTA and exposed for 5 s to 40 uM NMDA followed by a 0.4 s flash of UV light (U). Baseline [Ca2+]i was ~ 100 nM, following NMDA application [Ca2+]; reached ~ 1000 nM and following flash photolysis of NP-EGTA [Ca2+]; reached ~ 900 nM. + CM O 1200 i 1000 800 H 600 -400 200 0 NMDA 0 2 4 46 post-photolysis at which time [Ca2+]i was already in the process of decaying back to baseline values. Comparison of Room Temperature vs. 37 °C rates of recovery Following flash photolysis of NP-EGTA, the resulting Ca2+decay curve was, in the majority of situations, best fit by a double exponential decay curve, a result consistent with the observations of Kennedy & Thomas (1995) and Mironov (1995). In order to improve the resolution of the double exponential and to minimize the effects of dye leakage, all experiments were performed at room temperature (22 °C; Zavoico and Cragoe, 1988; Kao, 1994). The average control rates of recovery (N=92; N=number of cells) were 14.06 ± 3.52 for the fast component and 72.44 ± 32.19 for the slow component. These values are similar to those found by Mironov (1995) and reflect the slow nature of calcium transients at room temperature. Experiments were also performed at 37 °C in order to establish that decay curves at physiological temperatures were similar to those obtained at 22 °C. At 37 °C, the Ca2+ decay curve was also best fit by a double exponential with both a fast and a slow component; however, the noticeable difference was the much faster recovery to baseline [Ca2+]; (Fig. 8 and Table 2). The following table lists the control values obtained at room temperature and 37 °C, two sets of values are shown, obtained on separate days, demonstrating the marked variability in decay rates between days. Despite this marked variability between days, within a single day there was no significant variability thus emphasizing the use of paired same-day controls obtained from sister-cultures. 47 Figure 8: Typical response seen at 37 °C. Mean response of a field of neurons (n=12) loaded with both fluo-3 and NP-EGTA. Following a 5 s exposure to 40 uM NMDA, resting Ca2+-levels increased and then decayed back to baseline levels within the next 200s. Following flash photolysis of NP-EGTA, Ca2+-levels increased and then decayed back to baseline levels at a much quicker rate than observed at room temperature. 49 Table 2: Comparison of Room Temperature vs. 37 °C rates of recovery FAST SLOW N T(S) S.E.M. T(S) S.E.M. Control-RT 13 7.39 0.16 87.32 2.75 Control-37 °C 9 2.28 0.14 20.32 . 1.32 Control-RT 5 10.91 0.80 37.45 2.96 Control-37 °C 16 4.35 0.31 26.36 0.69 III. Role of the PM-Na+/Ca2+ exchanger In neurons loaded with the pH sensitive fluorescent indicator BCECF, removal of external Na+ and replacement with either NMDG or Li+ (Figs. 9 and 10 respectively), resulted in a rapid fall in pH; from resting values of ~ 6.8 to values of-6.4 or less. For 15 neurons, normal resting pHj was 6.83 ± 0.04, following removal of all external Na+ (replaced by NMDG) pH; fell to statistically different levels of 6.38 ± 0.02 (p<0.001, paired r-test, Fig. 9), this effect lasted 20 minutes until 10 uM nigericin was added to the superfusate. In another field of 20 neurons, normal resting pHj was 6.79 ± 0.04, following removal of external Na+ (replaced by NMDG), pH; fell to 6.25 ± 0.03 (p<0.001, paired f-test, Fig. 9). With the addition of 5 mM TMA, pH; returned to 6.77 ± 0.02 which was not statistically different from the original resting pHi (paired Mest). Without correcting for changes in pHi, removal of all external Na+ (replaced by NMDG) resulted in a significant prolongation of the recovery of background corrected fluo-3 50 Figure 9: The effects of 0Na+-NMDG on pHj. Record from two separate fields of neurons obtained from sister cultures loaded with BCECF. The solid line represents a field of neurones (n=15) exposed to 0 Na+-NMDG for 27 minutes followed by a 15 minute exposure to 10 pM nigericin (*). Upon substitution of extracellular Na+ with NMDG, pEL falls from a resting value of 6.83 ± 0.04 to 6.38 ± 0.02. The dotted line represents a field of neurons (n=20) continuously exposed to 0 Na+- NMDG for 26 min; after 15 minutes, pFh fell to 6.25 ± 0.03 from its resting value of 6.79 + 0.04; at this point (A), 5 mM TMA was added to the medium which restored pEL to 6.77 ± 0.02 it was at this point, when pH; was restored to baseline values, where the uncage was performed in sister cultures loaded with fluo-3 and NP-EGTA; 10 uM nigericin was then superfiised (*) for calibration purposes. 52 Figure 10: The effects of 0Na+-Li+ on pHj. Mean response of a field of neurons (n=20) loaded with BCECF. Following removal of external Na+ (replaced by Li+) resting pH; fell from a value of 6.97 ± 0.03 to 6.35 ± 0.04, pH; recovered slowly over the course of the next 30 min, 10 uM nigericin was then superfused (*) for calibration purposes. Time (min) 54 fluorescence intensities to baseline values following photolysis of NP-EGTA (Fast, p<0.001; Slow, p<0.001; unpaired /-test, see Fig. 11 and Table 3). A slowing down of the initial fast recovery phase was particularly evident. However, restoring pH; to normal values eliminated this effect of 0 Na+. Under these conditions there was no evidence to support a role for the PM-Na+/Ca2+ exchanger in the recovery of background corrected fluo-3 fluorescent intensities to baseline values (Fast, N/S; Slow, N/S; unpaired /-test, Table 3). In 20 neurons, when Li+ was used to replace external Na+, we observed an immediate fall in pHj from resting levels of 6.97 ± 0.03 to 6.30 ± 0.04 (pO.OOl, paired r-test), pH; levels then began to slowly recover (Fig. 10). We therefore examined the recovery of fluo-3 fluorescence following flash photolysis of NP-EGTA at four different time intervals after the onset of superfusion with Li+-containing solutions. After 4 and 12 minutes, we observed a significant prolongation in the rate of recovery (4 min-Fast-p<0.05, Slow-p<0.05; 12 min- Fast-p<0.05, Slow-p<0.001, unpaired /-test), but with longer superfusions this effect declined and by 40 minutes there was no significant difference in the rates of recovery when compared to control values obtained in sister cultures (Fast-N/S, Slow-N/S, unpaired /-test; see Table 3). IV. Role of mitochondria In 21 neurons, when the protonophore CCCP (2 uM) was used to inhibit mitochondrial uptake of Ca2+ in neurons loaded with BCECF, within 4 minutes we observed a fall in pH; from resting levels of 6.86 ± 0.04 to statistically different levels of 6.66 ± 0.04 (pO.OOl; paired /-test, Fig. 12), this effect lasted for 15 minutes until 10 u.M nigericin was added to the superfusate. In 55 Figure 11: Background subtracted normalized data showing the influence of the PM-Na+/Ca2+ exchanger on Ca2+-homeostasis. Normalized mean response of three independent fields of neurons from sister cultures loaded with both fluo-3 and NP-EGTA. When Na+ was replaced by NMDG (•) (n=ll), the rate of recovery back to baseline values was much slower than that of the controls (o) (n=8). However if photolysis was performed 1 min after restoring pH; to baseline values with 5 mM TMA, the resulting decay curve (A) (n=13) was virtually identical to that seen in controls (o). (•, o, and A represent actual data points while the solid line represents the best fit double exponential decay curve.) 56 57 Figure 12: The effects of CCCP on resting pHi. Mean response of a field of neurons (n=21) loaded with BCECF. When 2 uM CCCP was superfused, pH; fell from 6.86 ± 0.04 to 6.66 ± 0.04. Nigericin (10 uM) was then superfused (*) for calibration purposes. Time (min) 59 another field of 25 neurons, resting pH; levels of 6.85 ± 0.04 fell, after 2 minutes, to statistically different levels of 6.67 ± 0.03 following superfusion of 2 uM CCCP (pO.OOl; paired /-test, Fig. 13). Within 1 minute of the addition of 2 mM TMA to the superfusion medium pH; was restored to 6.84 ± 0.03, which was not statistically different from the original resting levels obtained prior to superfusion with CCCP (paired /-test). This procedure was repeated using sister cultures loaded with fluo-3 and NP-EGTA to assess the effect of CCCP on the recovery of background corrected fluo-3 fluorescent intensities to baseline values following photolysis of NP-EGTA. Superfusion of CCCP alone produced an apparent rise in background corrected fluo-3 fluorescence (Fig. 14) which was rapidly eliminated when TMA was added to the medium (Fig. 15). Uncaging in CCCP resulted in a significantly slower recovery to baseline values (Fast, pO.OOl; Slow, p<0.05; unpaired /-test, see Table 3). When the pH; was restored to resting levels, uncaging (after 60 s of superfusion in TMA, 300 s in CCCP) resulted in a release of Ca2+ with a much slower recovery to baseline values well above prior resting levels (Fast, pO.05; Slow, p<0.05; unpaired /-test). This effect of CCCP is evident in the normalized data shown in Fig. 16 and Table 3. Compared to control rates of recovery, CCCP treated neurons, with normal pHi restored by TMA, had significantly slower rates of recovery (Fig. 16). In view of the fact that CCCP will deplete ATP levels in addition to its action on mitochondrial Ca2+-uptake, we performed additional experiments in the presence of 20 uM of the ATP-synthase inhibitor oligomycin. This served to inhibit the reverse activity of the ATP-synthase resulting from the collapse of the mitochondrial proton gradient. ATP/ADP levels will then be transiently maintained (most likely due to glycolysis) and any changes in Ca2+-extrusion rates can be attributed to inhibition of mitochondrial Ca2+-uptake rather than ATP depletion (Budd & Nicholls, 1996). Under these conditions we still observed a significant prolongation 60 Figure 13: The effects of CCCP and CCCP + TMA on pEL. Mean response of a field of neurons (n=25) loaded with BCECF. When 2 pM CCCP was superfused, resting pH; fell from 6.85 ± 0.04 to 6.67 ± 0.03. Within 1 min of superfusion with 2 mM TMA pH; was restored to its resting value of 6.85 ± 0.03, at this point the uncage was performed ensuring that pFh was at resting values. Nigericin (10 pM) was then superfused (*) for calibration purposes. 62 Figure 14: The effects of CCCP on resting Ca -levels. Mean response of a field of neurons (n=12) loaded with fluo-3 and NP-EGTA. Following a 5 s application of 40 uM NMDA, Ca2+-levels increased before decaying back to baseline levels. Following superfusion of 2 pM CCCP, resting Ca2+-levels increased and remained at this elevated level for the remainder of the experiment (700 s). 260 n 0 200 400 600 800 1000 1200 1400 Time (s) 64 Figure 15: The effects of CCCP and CCCP + TMA on Ca -levels Mean response of a field of neurons (n=16) loaded with fluo-3 and NP-EGTA. Following a 5 s application of 40 pM NMDA, 2 uM CCCP was added to the superfusate. After 240 s in 2 pM CCCP, 2 mM TMA was added to the superfusate at which point Ca2+-levels began to decay back to baseline values. Following 60 s of superfusion in TMA, 300 s in CCCP an uncage was performed (U). Background subtracted fluo-3 fluorescence values then decayed back to values similar to those seen immediately prior to photolysis. Time (s) 66 Figure 16: Background subtracted normalized data showing the influence of CCCP on Ca2+-homeostasis. Normalized mean response of two independent fields of neurons from sister cultures loaded with both fluo-3 and NP-EGTA. When 2 uM CCCP was superfused and pH; was corrected for by the addition of 2 mM TMA (•) (n=7) the resulting decay curve was significantly slower than controls (o) (n=6). (• and o represent actual data points while the solid line represents the best fit double exponential decay curve.) 67 Time (s) 68 (Fast-p<0.001, Slow- p<0.001, unpaired t-test) in the rate of recovery from a flash-photolysis-induced increase in [Ca2+]i (see Table 3). We also inhibited mitochondrial electron transport by superfusion of 5 pM rotenone (Budd and Nicholls, 1996). In 32 neurons, resting pH; of 6.92 ± 0.02 remained at non-significantly different levels of 6.91 + 0.02 after two minutes of rotenone superfusion (Fig. 17; paired 7-test), at this point the uncage was performed. Unlike the effect of CCCP, rotenone alone had no effect on the baseline [Ca2+]i and following flash photolysis of NP-EGTA, the [Ca2+]i returned close to original resting levels (Fig. 18). Under these conditions we observed a significant prolongation in both the fast and slow component of the recovery (Fast-p<0.05, Slow-p<0.001, unpaired t-test) which is evident in the normalized data shown in Fig. 19 and Table 3. V. RoleoftheER-ATPase In an attempt to assess the influence of Ca2+-uptake into the ER following increases in [Ca2+]i we superfused either 2 \iM thapsigargin or 10 pM CPA in order to block the ER-ATPase (Mironov, Usachev and Lux, 1993; Bleakman, Roback, Wainer, Miller and Harrison, 1993; Seidler, Jona, Vegh, and Martonosi, 1989) or 20 pM ryanodine which, at this concentration, is sufficient to block ryanodine sensitive Ca2+ channels (Kiedrowski and Costa, 1995). Superfusion of thapsigargin, CPA, and ryanodine alone did not produce any significant changes in pHi (Fig. 20, 21, 22). We found the results of these experiments to be highly variable and in some cases we even observed a small increase in the rate of recovery of fluo-3 fluorescence. However, we did not observe any consistent reduction in the rate of recoveries of either the fast or slow components. 69 Figure 17: The effect of rotenone on pHj. Mean response of a field of neurons (n=32) loaded with BCECF. Following the addition of 5 uM rotenone to the superfusate, pH; began to slowly decrease. At (A) the uncage was performed to ensure that pHi was at levels that were not statistically different from resting levels. Following 10 min of superfusion with rotenone, nigericin (10 uM) was added to the superfusate (*). 7.10 7.05 J 7.00 J 6.95 6.90 6.85 Rotenone 6.80 0 5 10 15 20 Time (min) 71 Figure 18: The effect of rotenone on Ca -levels. Mean response of a field of neurons (n=10) loaded with both fluo-3 and NP-EGTA. Following a 5 s application of 40 uM NMDA, Ca2+-levels returned to baseline values. At this point rotenone (5 uM) was added to the superfusate and 2 min later an uncage was performed (U). During rotenone superfusion, baseline intensities did not change and flash photolysis of NP-EGTA produced a large transient increase in intensity which returned to levels close to the initial baseline values. 72 Time (s) 73 Figure 19: Background subtracted normalized data showing the influence of rotenone on Ca2+-homeostasis. Normalized mean response of two independent fields of neurons from sister cultures loaded with both fluo-3 and NP-EGTA. When 5 uM rotenone was superfused (•) (n=10) the resulting decay curve was significantly slower than controls (o) (n=16). (• and o represent actual data points while the solid line represents the best fit double exponential decay curve.) 74 Time (s) 75 Figure 20: The effect of thapsigargin on pHi. Mean response of a field of neurons (n=12) loaded with BCECF. pH; was unaffected by the addition of 2 pM thapsigargin to the superfusate. Nigericin (10 pM) was added to the superfusate for calibration purposes (*). 7.6 7.5 7.4 7.3 7.2 ] 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 Thapsigargin 0 10 15 20 Time (min) 77 Figure 21: The effect of CPA on pHt. Mean response of a field of neurons (n=13) loaded with BCECF. CPA (10 pM) was added to the superfusate for 10 minutes, during this time pEL was unaffected. Nigericin (10 pM) was added to the superfusate for calibration purposes (*). Time (min) 79 Figure 22: The effect of ryanodine on pHi. Mean response of a field of neurons (n=29) loaded with BCECF. Ryanodine (20 pM) was added to the superfusate for 10 minutes, during this time pH; was unaffected. Nigericin (10 pM) was added to the superfusate for calibration purposes (*). 7.5 -, Ryanodine 7.4 -7.3 -7.2 7.1 -7.0 -6.9 -0 5 10 15 20 Time (min) 6.7 81 In view of the detrimental effects that changes in pH; have on Ca """-homeostatic mechanism, attempts were made to 'clamp pH;' using the K+ ionophore nigericin (Fig. 23). In 19 neurons, following application of a high K+/nigericin solution, Ca2+ levels increased as a result of plasma membrane depolarization and the resulting activation of VGCC. Following recovery to baseline [Ca2+];, 40 uM NMDA was superfused for 5 s. In this situation, the observed responses were drastically different to those typically seen during NMDA application. VI. Summary and statistical analysis of data The following table lists the values obtained when individual Ca2+-homeostatic mechanisms were inhibited prior to flash photolysis of NP-EGTA, the table lists the time constants for both the fast and slow components along with the statistical significance (students two-tailed unpaired f-test) between experiments and controls: ] 82 Table 3: Statistical analysis of potential calcium homeostatic mechanisms FAST SLOW N x(s) S.E.M. SIG. x(s) S.E.M. SIG. Control 33 15.82 1.37 57.12 4.20 0 Na+-NMDG 7 43.20 3.13 p<0.001 78.73 3.35 p<0.001 0 Na+-NMDG + TMA 26 13.55 0.64 NS 66.70 5.62 NS Control 10 7.49 1.03 27.86 1.69 0 Na+-Li+ (4 min) 3 24.69 2.83 P<0.05 74.58 9.71 p<0.05 0 Na+-Li+ (12 min) 6 23.24 5.34 PO.05 69.98 6.79 p<0.001 0 Na+-Li+ (25 min) 3 13.53 6.10 NS 44.07 2.45 p<0.05 0 Na+-Li+ (40 min) 2 11.15 1.41 NS 37.73 2.20 NS Control 22 12.97 0.57 100.21 3.92 CCCP 19 25.30 1.99 p<0.001 115.34 7.20 p<0.05 Control 11 15.62 2.47 62.22 1.94 CCCP + TMA 3 36.63 4.86 PO.05 102.85 7.93 p<0.05 CCCP + TMA + oligomycin 5 43.53 3.95 p<0.001 89.97 3.64 p<0.001 Control 16 14.94 0.92 79.93 1.58 Rotenone 10 20.34 1.33 P<0.05 168.46 4.55 pO.001 83 Figure 23: The effect of clamping pHj using high K^-nigericin during NMDA application. Individual responses of 19 neurons loaded with both fluo-3 and NP-EGTA. A 5 s application of 40 u.M NMDA produced a typical profile (see Fig. 1, 2, 3 for comparison) which subsequently decayed back to baseline values within 300 s. A high K+-nigericin containing solution was then superfused for the remainder of the experiment. Following the depolarization of the plasma membrane and the subsequent entry of Ca2+ via voltage gated Ca2+-channels, Ca2+-levels decayed back to near resting levels. At this point 40 uM was applied for 5 s. The resulting responses were unlike any previously seen and many of the neurons did not recovery to baseline values during the next 300 s. 84 0 -] 1 1 1 P 0 200 400 600 800 1000 1200 1400 1600 Time (s) 85 DISCUSSION The aim of this study was, firstly, to develop a procedure to increase [Ca2+]; using the caged Ca2+-compound NP-EGTA, and secondly, to examine the potential Ca2+-homeostatic mechanisms responsible for reducing background subtracted fluo-3 levels back to baseline values following flash photolysis of NP-EGTA. Because of the interrelationship between [Ca2+]; and pH;, and the potential effects of experimental manipulations on the latter, this study sought to maintain pH; at a constant level thereby eliminating any of the effects that changes in pHi have on Ca2+-homeostatic mechanisms and fluorescent indicator dyes. The effects of drugs or ion substitutions on pET, were compensated for by the addition of the appropriate amount of the weak base TMA determined using the pH sensitive dye BCECF in sister cultures. Of the Ca2+-homeostatic mechanisms examined, only the inhibition of mitochondrial Ca2+-uptake, by the protonophore CCCP and the electron transport chain inhibitor rotenone, significantly reduced the rate of recovery from Ca2+-loads induced by flash photolysis of NP-EGTA. Inhibition of all other potential mechanisms (PM-Na+/Ca2+ exchanger, PM-ATPase or ER-ATPase) failed to have any significant effect on the rate of recovery of Ca2+-levels providing that pHj was either maintained or restored to normal baseline values with the weak base TMA. Loading with NP-EGTA andfluo-3 In order to induce increases in [Ca2+]; and record the subsequent Ca2+-decay curve, a method had to be developed which allowed for the co-loading of appropriate amounts of the caged Ca2+-compound NP-EGTA and fluorescent indicator fluo-3. In order to determine the correct levels of fluo-3 to allow for both baseline and peak [Ca2+]; determination with minimal 86 influences on the Ca2+ decay curve, many empirical trials were performed with various loading times and concentrations (see Table 1). As fluo-3 has a relatively high Kd for Ca2+ (~ 80 nM), low levels could not be used as the signal from cells with low resting [Ca2+]i would be undetectable. Similarly, high levels were not suitable as firstly, the mobile Ca2+-buffering capacity of the cytosol would be altered thereby influencing the kinetics of the Ca2+-decay curve and secondly, the fluo-3 fluorescence signal following photolysis would most likely saturate the camera thereby misjudging peak [Ca2+]i along with the initial portions of the Ca2+-decay curve (Ellis-Davies and Kaplan, 1994; Nerbonne, 1996). Loading parameters were therefore set at a level such that the neuronal Ca2+ buffering capacity was minimized while at the same time Ca2+-levels were detectable in all neurons, even those with low resting [Ca2+];. Similar to the effects of fluo-3 on the neuronal calcium buffering capacity, unphotolyzed NP-EGTA, as discussed by Lamb and Stephenson (1995), may rebind Ca2+ thereby influencing the kinetics of the Ca2+-decay curve; this problem will only be solved with the creation of caged Ca2+-compounds with much higher affinities for Ca2+ (in the range of 1-10 nM) such that the majority of the chelator is bound to Ca2+ prior to photolysis (Nerbonne, 1996). In order to minimize this effect of NP-EGTA, the loading parameters were set at a level such that, firstly, following flash photolysis, significant increases in intracellular Ca2+-levels were obtained, but secondly, that following flash photolysis the majority of the NP-EGTA present within the cytosol would be photolyzed thereby minimizing the influences of unphotolyzed NP-EGTA on the mobile Ca2+-buffering capacity of the cytosol. This was partially achieved with the use of a 0.4 s flash of UV light despite the fact that the release of Ca2+ from NP-EGTA occurs within 200 us (Nerbonne, 1996). By markedly increasing the required photolysis time, it was possible to ensure that the majority of the NP-EGTA within each neuron was photolyzed and, as a result, 87 could not bind with Ca2+ and thereby influence the mobile Ca2+-buffer capacity of the cytosol (see Fig. 2). Empirical trials were also performed in order to determine the appropriate levels of NP-EGTA needed to produce moderate increases in [Ca2+]i while at the same time minimizing the influences of NP-EGTA on the Ca2+-buffering capacity of the cytoplasm. If high concentrations of NP-EGTA were used for loading, the increase in [Ca2+]i following photolysis greatly exceeded the peak [Ca2+]; reached during a 5 s application of 40 pM NMDA, suggesting that this increase in [Ca2+]i was excessive in nature (data not shown). Determination of the appropriate loading conditions was further complicated by the relationship between caged compounds and fluorescent indicator dyes; the sensitivity of the fluorescent dye is changed depending on the concentration of the caged compound used and the ratio of the caged compound to fluorescent indicator (Zucker, 1992). As a result the relative amounts of fluo-3 and NP-EGTA were determined empirically to account for this phenomenon. The final loading concentrations of fluo-3-AM and NP-EGTA-AM were 3.1 p:M and 8 pM respectively. Loading was for 1 hour at room temperature and photolysis was induced by a 0.4 s flash of unfiltered UV light. NP-EGTA as a method for increasing [Ca2+Ji There are a number of properties of caged Ca2+-compounds which have to be taken into consideration when they are used in an intracellular environment. These include their sensitivity to pH, their affinity for Ca2+ and selectivity of Ca2+-binding with respect to other ions, especially Mg2+, the relative binding of the native molecules with respect to their photolysis products, and the quantum yield for Ca2+ release (i.e. the proportion of caged Ca2+-compound photolysed by UV light). DM-nitrophen, a derivative of the Ca2+-chelator EDTA, has a high affinity for Ca2+, a 88 high quantum yield (0.18), and, following flash photolysis, the resulting products have a 6.0 x 105 fold decrease in the affinity for Ca2+. However, it shares with its parent molecule EDTA, a high sensitivity to changes in pH and a high affinity for Mg2+. Nitr-5, a derivative of B APT A, is much more selective for Ca2+ over Mg2+, is relatively insensitive to changes in pH, but has a relatively low affinity for Ca2+, a low quantum yield (0.02), and only a 42-fold decrease in the affinity of the photolysis products for Ca2+. When using nitr-5 to increase [Ca2+];, it was apparent that significant increases in [Ca2+]i were reached only when high levels of [Ca2+]i were present immediately preceding photolysis (see Fig. 4); this is most likely due to the relatively high Kd of nitr-5 for Ca2+. As a result it was necessary to utilize a caged Ca2+-compound with much higher affinities for Ca2+such that a larger percentage of the compound would be bound to Ca2+ prior to photolysis thereby negating the post-photolysis influences on the mobile Ca2+-buffering capacity of the cytosol. NP-EGTA, a relatively new caged Ca2+ compound, meets a number of important criteria not previously attained by nitr-5 or DM-nitrophen (Ellis-Davies and Kaplan, 1994; Nerbonne, 1996). NP-EGTA has a much lower affinity for Mg2+, its flash photolysis products have a 1.2 x 104 fold decrease in affinity for Ca2+ and it has a high quantum yield (0.23). In addition, because it has a Kd close to the resting [Ca2+]; of most cells (~ 80 nM in our neurons), at least 50% of the molecules would be in the Ca2+-bound form thereby minimizing the post-photolysis effects on the mobile Ca2+-buffering capacity of the cytosol. These properties have proved to be useful under physiological conditions, and flash photolysis of NP-EGTA has been previously used to induce successfully the endocytosis of secretory granules in mouse pancreatic P-cells (Eliasson et al, 1996), contract chemically skinned skeletal muscle fibres in rabbit (Ellis-Davies and Kaplan, 1994), and induce exocytosis in rat pituitary melanotrophs (Parsons et al, 1996). 89 In our experiments we have used NP-EGTA to assess neuronal Ca2+-homeostasic mechanisms, and for this purpose there are many potential advantages over more traditional methods of increasing [Ca2+]; such as the activation of VGCC or LGCC. Firstly, NP-EGTA can be loaded as an AM-ester and, after crossing the plasma membrane, is cleaved into free NP-EGTA which subsequently binds Ca2+ and equilibrates with the prevailing free Ca2+ concentration within the neuron. This is a useful property for the study of multiple cells simultaneously, especially when the preparation consists of multiple phenotypes as is the case with primary cultures of rat hippocampal neurons. Secondly, the release of Ca2+ as a result of photolysis is extremely rapid (< 200 ps) and the recovery following flash photolysis can be measured in the absence of any further influx (Nerbonne, 1996). Thirdly, NP-EGTA is freely diffusible and distributes evenly within the cytoplasm. The release of Ca2+ will therefore be uniform throughout the cytoplasm and large Ca2+-gradients typically seen when increasing [Ca2+]; via traditional methods (VGCC or LGCC) will be avoided. Fourthly, increasing Ca2+ via VGCCs and LGCCs results in relatively slow increases in [Ca2+]; with continued Ca2+-influx occurring at the same time as neuronal Ca2+-homeostasic mechanisms being activated, an effect not seen with the use of NP-EGTA. Additionally, as previously mentioned, depending on the method used to increase [Ca2+]j3 different Ca2+-homeostatic mechanisms may be activated (White and Reynolds, 1995). In addition to these advantages, with the use of NP-EGTA the 'nature' of the stimuli is unchanged between experiments and controls, and the Ca2+-load will be very similar in both situations. For example, following replacement of all external-Na+ with NMDG, two changes become apparent; firstly, pHj decreases and secondly, the normal entry of Na+ through LGCC, following application of NMDA, is absent. As a result, the Ca2+-load induced in Na+-free 90 conditions differs from control situations not only as a result of the influences of pHi on intracellular constituents but also as a result of the lack of Na+ influx through VGCC or LGCC. This Na+ influx will, in turn, influence [Ca2+]j by way of a destabilization of Ca2+-homeostatic mechanisms, in particular the PM-Na+/Ca2+exchanger (Kiedrowski et al, 1994). In an attempt to minimize this influence, White and Reynolds (1995) applied 0 Na+ solutions immediately following the application of high K+ or glutamate but the timing involved in this procedure is virtually impossible to coordinate and, as a result, would not be reproducible between experiments. Despite NP-EGTA being a marked improvement over other methods of increasing [Ca2+]j, there are some potential flaws in its use as a means of examining Ca2+-homeostatic mechanisms. The homogeneous increase in [Ca2+];, while allowing for an accurate assessment of Ca2+-homeostatic mechanisms, is substantially different from the spatio-temporally graded signal that a neuron normally receives on activation of VGCC or LGCC. Those regions which receive higher amounts of Ca2+-entry typically contain higher proportions of Ca2+-homeostatic mechanisms as compared with regions with lower influx and, as a result, not all neuronal regions will be equipped to handle a global increase in [Ca2+]; (Tymianski et al, 1993; Rizzuto et al, 1994; Reuter and Porzig, 1995; Stauffer et al, 1997). The preferential location of the Na+/Ca2+ exchanger in the synaptic regions of dendrites in hippocampal neurons is one example (Reuter and Porzig, 1995; Luther et al, 1992). A second potential flaw is that the flash of light used for photolysis of NP-EGTA may not be constant between experiments, the intensity will change as the mercury arc-lamp ages and is replaced. These changes will influence the amount of Ca2+ released following photolysis and may not allow for reproducible results, particularly when comparing results obtained with a new rather than an old arc-lamp. Also, NP-EGTA, like its 91 parent molecule EGTA, is pH sensitive, and a normal pH; range must be maintained for NP-EGTA to be useful (Kao, 1994). Finally, the Ca2+-buffering capacity of the cytosol will be influenced by both NP-EGTA and fluo-3. This problem is inherent with the use of all caged Ca2+-compounds and fluorescent indicator dyes, but, by using the lowest concentration possible of both these compounds while still maintaining the capability to record accurate signals, even in those neurons with low resting [Ca2+];, these influences were minimized. While recognizing the influence of both NP-EGTA and fluo-3 on intra-neuronal Ca2+, the use of day-matched controls for each experimental condition ensures that these effects are constant and other Ca2+-homeostatic mechanisms can be measured in their presence. The other problems associated with the use of NP-EGTA, pH sensitivity and bulb aging, were both minimized by maintaining pH; at a constant level with the use of TMA and by performing day and coverslip matched controls. In order to obtain an accurate assessment of both baseline [Ca2+]i and [Ca2+]; following flash photolysis of NP-EGTA, the ratiometric dye fura-2 was used. Resting [Ca2+]i's were found to be ~ 80 nm, consistent with values previously obtained in similar cultures (Abdel-Hamid and Baimbridge-submitted; Abdel-Hamid and Baimbridge-in press), and following photolysis, the peak [Ca2+]j reached values ~ 800 nm, depending on the duration of the flash. Unfortunately, several problems exist with the use of fura-2 and NP-EGTA concurrently. Firstly, fura-2 is excited and bleached by the same wavelengths of light used to photolyse NP-EGTA and, conversely, the excitation of fura-2, may increase [Ca2+]; by releasing Ca2+ from NP-EGTA (Zucker, 1992; Adams and Tsien, 1993). This continuous release of Ca2+ when measuring [Ca2+]; with fura-2, may explain the minimal increases in [Ca2+]i seen following flash photolysis of NP-EGTA, a large percentage of the Ca2+ held by NP-EGTA may have already been released 92 (see Fig 5). Similarly, resting [Ca2+]i may have been artificially raised by the continuous release of Ca2+ from NP-EGTA during fura-2 excitation. In order to demonstrate that significant increases in [Ca2+]; could be reached with the concurrent use of fura-2 and NP-EGTA, neurons (loaded with fura-2 and NP-EGTA) were exposed to UV light for 0.5 s. This produced larger increases in [Ca2+];, following photolysis, than were previously seen using a 0.4 s flash and further demonstrates that when imaging Ca2+, using fura-2, in neurons loaded with NP-EGTA, the excitation wavelengths used for fura-2 release NP-EGTA from its cage, thereby decreasing the amount of NP-EGTA available for photolysis. As a result of the problems associated with the concurrent use of fura-2 and NP-EGTA, fura-red, which is excited at much longer wavelengths, was also utilized. This permitted the photolysis of NP-EGTA without any adverse effects on fura-red or, conversely, the excitation of fura-red without releasing Ca2+ from NP-EGTA. Using fura-Red, baseline [Ca2+]i were found to be ~ 100 nM consistent with those obtained previously in identical neuronal cultures and peak [Ca2+]j were found to be ~ 900 nM consistent with the physiological increases seen during a 5 s application of 20 uM NMDA (Abdel-Hamid and Baimbridge-submitted). For the majority of experiments, the single wavelength dye fluo-3 was used to assess [Ca2+]i. Reasons for using fluo-3 include, firstly, the excitation wavelength used for fluo-3 does not photolyze NP-EGTA, secondly, the marked lack of an effect that caged Ca2+-compounds have on fluo-3 as opposed to ratiometric dyes (Zucker et al, 1992; Hadley et al, 1993), and thirdly, the much higher data acquisition rate of fluo-3 over ratiometric dyes allowing for much more accurate assessments of the kinetics of Ca2+-decay curves. 93 Rates of recovery of [Ca2+]i at 22 ° and 37° C Decay curves recorded at 37 °C could always be resolved into double exponentials with fast and slow components which were similar but faster than those seen at room temperature, most likely reflecting increased enzymatic reaction speeds at higher temperatures. As temperature increases, the relative speed of a particular Ca2+-homeostatic process will increase depending on its Qio value (the fold increase in rate for a 10 °C increase in temperature). As a result it might have been possible to assign either the fast or the slow component to a particular homeostatic mechanism if the decrease in decay rates of one of the components, with increasing temperature, could be correlated to the Qio value of a particular Ca2+-homeostatic mechanism. For example it is known that the rate constant for mitochondrial Ca2+-uptake increases by 60 % from 22 - 37 °C (Marengo et al., 1997). However, numerous other factors are influenced by changes in temperature. For example, the Kd for Ca2+ of EGTA and BAPTA, the two principal compounds used to produce fluorescent Ca2+-indicator dyes, are very temperature sensitive (Harrison and Bers, 1987). Additionally, the amount of Ca2+ released from NP-EGTA following flash photolysis would be different at room temperature versus 37 °C as a result of the temperature sensitivity of the binding of NP-EGTA to Ca2+, and the kinetics of Ca2+-transients has been shown to be altered at different temperatures (Shuttleworth and Thompson, 1991). As a result of the multitude of factors influenced by temperature, we have not attempted to assign either the fast or the slow component to a particular Ca2+-homeostatic mechanism on the basis of differences in recovery rates recorded at different temperatures. Nonetheless, we can conclude that Ca2+-decay curves are best-fit by a double exponential at both 37 °C and room temperature, and that the interpretation of our data collected mostly at room temperature is relevant to mechanisms active at 37 °C. 94 The influence of pH on Ca2+-homeostatic mechansims Intracellular pH (pFQ in the majority of cell types is kept constant within the range of 6.8 to 7.2 (Putnam, 1995). Any prolonged deviations from this range are known to result in cellular toxicity and/or cellular death (Kraig et al, 1987). In neurons, changes in pH; have been shown to affect the activity of many enzymes and ion channels, the activities of VGCC and LGCC and the excitability of neurons (Chesler and Kaila, 1992). Additionally, cytoskeletal elements (cell shape and motility), cell-cell coupling, and membrane conductance are all dependent upon pH; (Putnam, 1995). Of particular importance to the present study is the influences of pH; on Ca2+-regulatory mechanisms within neurons. For example, lowering pH; inhibits the PM-ATPase (Carafoli, 1987), decreases the affinity of Ca2+-binding proteins for Ca2+ (Ingersoll and Wasserman, 1971) and decreases mitochondrial Ca2+-uptake (Gambassi et al, 1993). Additionally, fluo-3, the fluorescent indicator dye used in this study, is very sensitive to changes in pHj and its Kd for Ca2+ increases significantly (i.e. becomes less sensitive) as acidity increases (Martinez-Zaguilan et al, 1996). Furthermore, as previously demonstrated by Koch and Barish (1994), intracellular acidification alone resulted in a greater than two-fold increase in the time required for [Ca2+]; to return to baseline levels. In many previous reports describing the Ca2+-homeostatic mechanisms of living cells, the influences of ion substitutions or drugs on pHi have been overlooked. For example, a common experimental method for inhibiting the Na+/Ca2+ exchanger involves replacing all external Na+ with either Li+ or NMDG. On the basis of these experiments it has been concluded that the Na+/Ca2+ exchanger contributes to the restoration of Ca2+ levels back to baseline values following relatively long (10-15 s) exposures to either high K+ (Mironov et al, 1993; Friel and 95 Tsien, 1994) or 3 pM glutamate (White and Reynolds, 1995). Our experiments clearly demonstrate one of the problems with this interpretation is that removing external Na+ results in an intracellular acidification due to the inhibition of the Na+/H+ exchanger, which is an important acid extrusion mechanism in central neurons, exchanging one intracellular H+ with one extracellular Na+ (Schlue and Dorner, 1992; Koch and Barish, 1994; Baxter and Church, 1996; Bevensee etai, 1996). White and Reynolds (1995) suggested that replacement of Na+ with Li+ would not reduce pHj, but, as we clearly show, this is not the case in our neuronal preparations. The effect of Li+ substitution on pHj are in fact time dependent. Initially there is a substantial fall in pHj but, unlike substitution with NMDG, the pH; slowly rises over a period of 30 minutes (Fig. 10). This delayed recovery of pH; is a result of Li+ being able to substitute for Na+ in the Na+/Hf exchanger (Aronson, 1985). Additionally, White and Reynolds (1995) assumed that by adding HCO3" to a Na+-free media (Na+ replaced by NMDG) pHj would be maintained due to the activity of the HCO37CI" exchanger, unfortunately they provided no evidence concerning the importance of the HCO37CI" exchanger in the control of pH; in their neuronal cultures. Further evidence demonstrating the lack of concern over pHj levels comes from experiments by Benham et al (1992) and Mironov (1995) who both superfused pH 8.8 solutions in order to inhibit the Ca2+-ATPase. While the Ca2+-ATPase would undoubtedly be inhibited, these high pH solutions would also increase pH;, influencing many intracellular constituents (fluorescent indicator dyes and Ca2+ homeostatic mechanisms), along with Ca2+-entry via NMDA channels, thereby producing uninterpretable and confusing results (Tang et al, 1990; Traynelis and Cull-Candy, 1990; Takaderae* al, 1992; Gottfried and Chesler, 1994). 96 We attempted to use the K+ ionophore nigericin to clamp pHj at a constant level in order to avoid the superfusion of TMA required to restore pH; to resting levels following ion substitution or drug superfusion. However, nigericin superfusion increased [Ca2+]; as a result of membrane depolarization due to the high K+ solution and the concurrent activation of VGCC (see Fig. 24). While the neurons were being continually superfused with high K+/nigericin, 40 uM NMDA was applied for 5 s. In this situation, the response observed was markedly different than that typically seen during NMDA application. In particular, in many neurons [Ca2+]; did not return back to baseline values and for those neurons where some recovery in [Ca2+]i did occur, the rate was greatly prolonged. The role of the PM-Na+/Ca2+ exchanger When the Na+/Ca2+ exchanger was inhibited, under conditions in which the concomitant effects of pHj are corrected for using TMA, we found no significant effect upon the rate of recovery of [Ca2+]; following its release from NP-EGTA. Similar results were found when Na+ was replaced by Li+ (Table 3). Our results are in agreement with those of Benham et al. (1989), Duchen et al (1990), Thayer and Miller (1990), Bleakman et al. (1993) and Stuenkel (1994), who all concluded that the PM-Na+/Ca2+ exchanger was not important in reducing Ca2+-loads in neurons. While the failure to take into account the effects on pH; may explain the opposite conclusion reached by Mironov et al. (1993) and White and Reynolds (1995), it is possible that the contribution of PM-Na+/Ca2+ exchange to Ca2+-homeostasis varies considerably depending upon the particular neuronal phenotype. Indeed, White and Reynolds (1995), using cultured rat forebrain neurons, reported considerable variability in the degree of dependence of the PM-Na+/Ca2+ exchanger in the recovery of an increase in [Ca2+]; in individual neurons resulting from glutamate stimulation. Amiloride and its analogues, currently the most specific inhibitors for the 97 Na+/Ca2+ exchanger, were not used as these compounds also inhibit the Na+/H+ exchanger and are fluorescent at wavelengths similar to those used in the excitation of fluo-3 (White and Reynolds, 1995). Experiments in which Na+ was replaced with Li+ were particularly revealing in that the effects of Li+ on pH; are in fact time dependent. Initially there is a substantial fall in pH; but, unlike substitution with NMDG, the pHj does slowly recover and this recovery is more rapid and complete if experiments are performed at 37°C (Baxter and Church, 1996). We observed an apparent reduction of the rate of recovery of [Ca2+]; following its release from NP-EGTA only when its effects upon pH; was also apparent and not when pHj had recovered. These experiments confirm the relatively unimportant role of the Na+/Ca2+ exchanger in the overall Ca -homeostasis of our neuronal preparations without the necessity of the use of TMA required to restore pFL when Na+ is replaced by NMDG. The role of mitochondria Ca2+ uptake into mitochondria occurs at the expense of ATP production. Mitochondria take up Ca2+ via a uniporter driven by the large electrochemical gradient across the mitochondrial inner membrane (Werth and Thayer, 1994). Ca2+ exits the mitochondria via a Na+/Ca2+ exchanger, and Na+ is then removed via a Na+/H+ exchanger, the net result being that two H+ ions are prevented from entering the mitochondria via the ATP-synthase resulting in a decrease in ATP production. This study demonstrates that mitochondria are the principal mechanism responsible for sequestering Ca2+ in neonatal rat hippocampal neurons. Using 2 pM of the protonophore CCCP, we found a significant prolongation of the rate of recovery to 98 baseline values following flash photolysis of NP-EGTA and this effect was still observed when the fall in pH; induced by CCCP was restored with TMA. Similar results were found when mitochondrial Ca2+-uptake was inhibited by superfusion of 5 uM of the respiratory chain inhibitor rotenone (see Table 3). The conclusion that mitochondrial Ca2+-uptake is a major source of Ca2+-sequestration is in agreement with Thayer and Miller (1990), Tatsumi and Katayama (1993), Stuenkel (1994), Werth and Thayer (1994) and White and Reynolds (1995). When using protonophores such as CCCP consideration must be given to their effect on intracellular ATP levels in addition to their effect on pEL. Protonophores are highly effective at collapsing the proton gradient across the mitochondrial inner membrane resulting in a reversal of the inner mitochondrial membrane ATP-synthase and the conversion of ATP to ADP and inorganic phosphate (Budd and Nicholls, 1996). In assessing Ca2+-homeostatic mechanisms it is therefore necessary to distinguish between a direct inhibition of mitochondrial Ca2+-uptake and an indirect inhibition of other potential mechanisms due to ATP depletion. Many groups have not accounted for ATP depletion following protonophore addition and as a result do not distinguish between longer recovery times to baseline levels as a result of inhibition of mitochondrial Ca2+-uptake or those resulting from the ensuing inhibition of an ATP-dependent Ca2+-homeostatic mechanism (Thayer and Miller, 1990; Wang and Thayer, 1994; Kiedrowski and Costa, 1995; White and Reynolds; 1995). Where others have simply measured ATP levels and, in doing so, have concluded that protonophores do not influence ATP stores (White and Reynolds, 1995) Budd and Nicholls (1996) demonstrated that it is the ATP/ADP ratio which is important for the functioning of many enzymes and not the absolute ATP level. In order to limit ATP depletion, we superfused our neuronal preparations with the ATP-synthase inhibitor oligomycin. When doing so, ATP/ADP levels are transiently maintained (most likely due to glycolysis) and any changes in Ca2+-extrusion rates can be attributed to inhibition of 99 mitochondrial Ca2+-uptake rather than ATP depletion (Budd and Nicholls, 1996). Under these conditions we still observed a marked prolongation of the recovery rate of background subtracted fluo-3 fluorescence levels following Ca2+-release from NP-EGTA (see Table 3). We have also considered the possibilities that protonophores may influence non-mitochondrial Ca2+-pools or may permeabilize the plasma membrane allowing H+ ions to move towards their Nernst electrochemical equilibrium, consequently influencing pH; or potentially depolarizing the plasma membrane thereby promoting Ca2+-entry via VGCC (Duchen, 1990; Jensen and Rehder, 1991; White and Reynolds, 1995; Budd and Nicholls, 1996b). Jensen and Rehder (1991) showed that in Helisoma neurons, FCCP induced an increase in [Ca2+]j that lasted indefinitely and was not influenced by earlier depletion of mitochondrial Ca2+-stores, and White and Reynolds (1995) showed that increases in [Ca2+];, in cultured rat cortical neurons, induced by FCCP application, were decreased when VGCC blockers were superfused. In our experiments, CCCP produced a small fall in pH; and only a transient increase in [Ca2+]; (see Fig. 15) suggesting that in neonatal rat hippocampal neurons, only minimal amounts of Ca2+ are released from the mitochondria. This transient increase in [Ca2+]; may originate from three other potential sources; firstly, inhibition of mitochondrial Ca2+-uptake, secondly, Ca2+-entry via VGCC secondary to a depolarization of the plasma membrane, and thirdly, a release of Ca2+ from non specific Ca2+ binding sites within the cytoplasm secondary to a decrease in pFL. Following the addition of TMA, this increase in [Ca2+]; reversed and declined (see Fig. 15) suggesting that the majority of the increase was pH related and occurred most likely as a result of protons dislodging Ca2+ from nonspecific intracellular binding sites and not as a result of further Ca2+-entry as a result of membrane depolarization (Meech and Thomas, 1977; Ou Yang et ai, 1995). This pH related increase in Ca2+ following superfusion of 2 pM CCCP is further strengthened by the fact 100 that when pH; was not corrected for, superfusion of 2 uM CCCP produced a long-lasting increase in [Ca2+]; (see Fig. 14). Similarly, following rotenone superfusion, which does not change pHj, baseline [Ca2+]i did not change (see Fig. 18) further strengthening our conclusion that rises in [Ca2+]i following CCCP superfusion which does change pH; levels, were in fact due to a reduction in pHj. We conclude from our observations that mitochondria are the major Ca2+-homeostatic mechanism available to cultured post-natal hippocampal neurons for the reduction of increases in [Ca2+]i to baseline values following flash photolysis of NP-EGTA. When inhibiting mitochondrial Ca2+-uptake with CCCP, [Ca2+]i never fully recovered to baseline levels most likely as a result of the effects of pH. On the other hand, when using rotenone to inhibit mitochondrial Ca2+ uptake, baseline values were restored in the majority of neurons measured. We believe that the principal reason for this discrepancy is the dramatically different effects of these two compounds on pH;. In view of the fact that, following inhibition of mitochondrial Ca2+-uptake, Ca2+-levels begin to decay towards baseline values, other mechanisms are most likely involved in the restoration of baseline Ca2+-levels. During control situations, when mitochondrial Ca2+-uptake is not inhibited, mitochondria sequester the majority of the Ca2+-increase induced by flash photolysis of NP-EGTA but, following inhibition of mitochondrial Ca2+-uptake, other previously unimportant mechanisms (Na+/Ca2+ exchanger, PM-ATPase, ER-ATPase) may begin to sequester Ca2+ in order to return Ca2+-levels to near baseline values. This explains the recovery of Ca2+-levels seen following flash photolysis of NP-EGTA in neurons in which mitochondrial Ca2+-uptake has been inhibited with CCCP or rotenone. The role of the ER 101 It has been reported that the caffeine sensitive Ca2+ stores may be substantially different in their magnitudes when comparing central (including hippocampal), and peripheral neurons (Shmigol et al, 1994). In the latter, caffeine results in a significant intracellular release of Ca2+ whereas in central neurons it is relatively ineffective. We used ryanodine, CPA and thapsigargin in attempts to inhibit the uptake of Ca2+ into intracellular stores such as the ER. The results of these experiments were quite variable and in some cases we even observed a small increase in the rate of recovery of fluo-3 fluorescence during application of these drugs. However, taken as a whole, we found no consistent evidence to suggest that the ER-ATPase was an important contributor to the homeostasis of Ca2+-loads induced by photolysis of NP-EGTA. A similarly negative conclusion resulted from experiments designed to determine the role of the PM-ATPase in Ca2+-buffering by inhibiting its activity with vanadate. Eosin, a potent and specific inhibitor of the PM-ATPase (Gatto et al, 1995), is fluorescent at the excitation wavelength used for fluo-3, and as a result it could not be used to assess the contributions of the PM-ATPase to Ca2+-homeostasis using our methods. i Conclusions, limitations and future directions From this study we conclude that following flash photolysis of NP-EGTA, the principal Ca2+-homeostatic mechanism responsible for reducing [Ca2+]; levels back to baseline values is mitochondrial Ca2+-uptake. Inhibition of all other potential Ca2+-homeostatic mechanisms did not influence the decay back to baseline values once normal pHi was either maintained or restored. There are however, several limitations associated with the above conclusions. Firstly, 102 [Ca2+]j was measured from the cell bodies of hippocampal neurons whereas many Ca2+-homeostatic mechanisms, for example the Na+/Ca2+ exchanger, have been shown to be preferentially localized to nerve terminals and presynaptic boutons (Luther et al, 1992; Reuter and Porzig, 1995). As a result inhibition of the Na+/Ca2+ exchange in the cell bodies may have had little effect as the highest proportion of PM-Na+/Ca2+ exchangers may have been found in other neuronal regions. Secondly, all experiments were performed at room temperature to improve the resolution of Ca2+-transients, decrease dye-leakage and decrease dye-compartmentalization. As the Qio values for Ca2+-homeostatic mechanism are very temperature sensitive, this decreased temperature may not have demonstrated the true contributions of Ca2+-2_|_ homeostatic mechanisms such as the Na+/Ca2+ exchanger, ER-ATPase or PM-ATPase to Ca -transients. Instead these potential homeostatic mechanism may have been operating at a lower rate as compared with those seen at physiological temperatures. This study found that mitochondrial Ca2+-uptake was the principal mechanism responsible for reducing [Ca2+]; back to baseline values. Using the fluorescent indicator dye rhod-2, which is selectively taken up by mitochondria, direct evidence for mitochondrial Ca2+-uptake could be obtained and is an attractive option for further experiments. Our conclusions are also limited by the lack of information regarding the absolute levels of [Ca2+]j, obtained by the photolysis of NP-EGTA, especially in relation to levels that might be achieved under more physiological conditions. It would therefore be useful to reexamine the effect of mitochondrial inhibitors following the release of variable amounts of Ca2+ obtained by varying the concentration of NP-EGTA within neurons. Under these conditions it might be possible to determine a threshold [Ca2+]j, beyond which mitochondrial sequestration of Ca2+ becomes significant. 103 Following inhibition of mitochondrial Ca2+-uptake, Ca2+-levels' decay towards initial levels suggesting that other homeostatic mechanisms may play a role in the restoration of baseline values. Experiments where multiple Ca2+-homeostatic mechanisms are simultaneously inhibited would demonstrate which of the potential Ca2+-homeostatic mechanism(s) may be operating during inhibition of mitochondrial Ca2+-uptake. 104 Abdel-Hamid, K.M. & Baimbridge, K.G. (1997). The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons. Neuroscience In Press. Adams, S.R. & Tsien, R.Y. (1993). Controlling cell chemistry with caged compounds. Annual Review of Physiology 55, 755-784. Ahmed, Z. & Connor, J.A. (1988). Calcium regulation by and buffer capacity of molluscan neurons during calcium transients. Cell Calcium 9, 57-69. Akerman, K.E.O., Wikstrom, M.K.F. & Saris, N.E. (1977). Effects of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria. Biochimica et Biophysica Acta 464, 287-294. Aronson, P.S. (1985). Kinetic properties of the plasma membrane Na+/H+ exchanger. Annual Review of Physiology 47, 545-560. Augustine, G.J., Charlton, M.P. & Smith, S.J. (1987). Calcium action in synaptic transmitter release. Annual Review of Neuroscience 10, 633-693. Baimbridge, K.G., Celio, M.R. & Rogers, J.H. (1992). Calcium-binding proteins in the nervous system. Trends in Neurosciences 15, 303-308. Bassani, J.W.M., Bassani, R.A. & Bers, D.M. (1995). Calibration of indo-1 and resting intracellular [Ca]; in intact rabbit cardiac myocytes. Biophysical Journal 68, 1453-1460. Baxter, K.A. & Church, J. (1996). Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. Journal of Physiology 493, 457-470. Bean, B.P. (1989). Classes of calcium channels in vertebrate cells. Annual Review of 105 Physiology 51, 367-384. Benham, CD., Evans, M.L. & McBain, C.J. (1992). Ca2+ efflux mechanisms following depolarization evoked calcium transients in cultured rat sensory neurones. Journal of Physiology 455, 567-583. Berridge, M.J. (1997). Elementary and global aspects of calcium signalling. Journal of Physiology 499, 291-306. Bevensee, M.O., Cummins, T.R., Haddad, G.G., Boron, W.F. & Boyarsky, G. (1996). pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. Journal of Physiology 494, 315-328. Blaustein M.P. (1988). Calcium transport and buffering in neurons. Trends in Neurosciences 11, 438-443. Blaustein, M.P. & Hodgkin, A.L. (1969). The effect of cyanide on the efflux of calcium from squid axons. Journal of Physiology 198, 46-48. Bleakman, D., Roback, J.D., Wainer, B.H., Miller, R.J. & Harrison, N.L. (1993). Calcium homeostasis in rat septal neurons in tissue culture. Brain Research 600, 257-267'. Brandl, C.J., Green, N.M., Korczak, B. & MacLennan, D.H. (1986). Two Ca2+-ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44, 597-607. Bright, G.R., Fisher, G.W., Rogowska, F. & Taylor, D.L. (1987). Fluorescence ratio imaging microscopy: Temporal and spatial measurements of cytoplasmic pH. Journal of Cell Biology 104, 1019-1033. Budd, S.L. & Nicholls, D.G. (1996). A Reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis. Journal of Neurochemistry 66, 403-411. Budd, S.L. & Nicholls, D.G. (1996b) Mitochondria, calcium regulation, and acute glutamate 106 excitotoxicity in cultured cerebellar granule cells. Journal of Neurochemistry 67, 2282-2291. Carafoli, E. (1987). Intracellular calcium homeostasis. Annual Review of Biochemistry 56, 395-433. Carafoli, E. (1991). The Calcium pumping ATPase of the plasma membrane. Annual Review of Physiology 53, 531-547. Caroni, P., Villani, F. & Carafoli, E. (1981). The cardiotoxic antibiotic doxorubicin inhibits theNa+/Ca2+ exchange of dog heart. FEBS Letters 130, 184-186. Chaillet, J.R. & Boron, W.F. (1985). Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. Journal of General Physiology 86, 765-94. Chard, P.S., Bleakman, J.D., Christakos, D., Fullmer, CS. & Miller, R.J. (1993). Calcium buffering properties of Calbindin D28k and parvalbumin in rat sensory neurones. Journal of Physiology 472,341-357. Chesler, M. & Kaila, K. (1992). Modulation of pH by neuronal activity. Trends in Neurosciences 15, 396-402. De Luca, H.F. & Engstrom, G.W. (1961). Calcium uptake by rat kidney mitochondria. Proceedings of the National Academy of Sciences of the United States of America 47, 1744-1750. Dipolo, R. & Beauge, L. (1979). Physiological Role of ATP-driven calcium pump in squid axon. Nature 278, 271-273. Duchen, M.R., Valdeolmillos, M., O'Neill, S.C. & Eisner, D.A. (1990). Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones. Journal of Physiology 424, 411-426. 107 Duchen, M.R. (1990). Effects of metabolic inhibition on the membrane-properties of isolated mouse primary sensory neurones. Journal of Physiology 424, 387-409. Dunham, E.T. & Glynn, I.M. (1961). Adenosine-triphosphatase activity and the active movements of alkali metal ions. Journal of Physiology 156, 274-293. Ebashi, S. & Lippman, F. (1962). Adenosine triphosphate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. Journal of Cell Biology 14, 389-400. Eliasson, L., Proks, P., Ammala, C, Ashcroft, F.M., Bokvist, K, Renstrom, E., Rorsman, P. & Smith, P.A. (1996). Endocytosis of secretory granules in mouse pancreatic P-cells evoked by transient elevation of cytosolic calcium. Journal of Physiology 493, 755-767. Ellis-Davies, G.C.R. & Kaplan, J.H. (1994). Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proceedings of the National Academy of Sciences of the United States of America 93, 187-191. Fabiato, A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. American Journal of Physiology 245, Cl-14. Farber, J.L. (1981). The role of calcium in cell death. Life Science 29, 1289-1295. Friel, D.D. & Tsien, R.W. (1994). An FCCP-sensitive Ca2+ store in bullfrog sympathetic neurons and its participation in stimulus-evoked changes in [Ca2+];. The Journal of Neuroscience 14, 4007-4024. Gambassi, G., Hansford, R.G, Sollott, S.J., Hogue, B.A., Lakatta, E.G. & Capogrossi, M.C. (1993). Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium. Journal of General Physiology 102, 575-597. 108 Gatto, C, Hale, C.C., Xu, W. & Milanick, M.A. (1995) Eosin, a potent inhibitor of the plasma membrane Ca pump, does not inhibit the Cardiac Na-Ca exchanger. Biochemistry 34, 965-972. Gleason, E., Borges, S. & Wilson, M. (1995). Electrogenic Na+-Ca2+ exchange clears Ca2+ loads from retinal amacrine cells in culture. The Journal of Neuroscience 15, 3612-3621. Goeger, D.E., Riley, R.T. & Dorner, J.W. (1988). Cyclopiazonic acid inhibition of the Ca2+-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochemical Pharamcology 37, 978-981. Gopinath, R.M. & Vincenzi, F.F. (1977). Phosphodiesterase protein activator mimics red blood cell cytoplasmatic activator of the (Ca2+ + Mg2+) ATPase. Biochemical and Biophysical Research Communications 77, 1203-1209. Gottfried, J.A. & Chesler, M. (1994). Endogenous H+ modulation of NMDA receptor-mediated EPSCs revealed by carbonic anhydrase inhibition in rat hippocampus. Journal of Physiology 478, 373-378. Grynkiewica, G., Poenie, M. & Tsien, R.Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. Gunter, T.E., Gunter, K.K., Sheu, S.S. & Gavin, C.E. (1994). Mitochondrial calcium transport: physiological and pathological relevance. American Journal of Physiology 267, C313-339. Gunter, T.E. & Pfeiffer, D.R. (1990). Mechanisms by which mitochondria transport calcium. American Journal of Physiology 258, C755-C786. Hadley, R.W., Kirby, M.S., Lederer, W.J. & Koa, J.P.Y. (1993). Does the use of DM-109 nitrophen, nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence. BiophysicalJournal 65, 2537-2546. Harrison, S.M. & Bers, D.M. (1987). The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-B APT A. Biochimica et Biophysica Acta 925, 13 3 -143. Hehl, S., Golard, A., & Hille, B. (1996). Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes. Cell Calcium 20, 515-524. Herrington, J., Park, Y.B., Babcock, D.F. & Hille, B. (1996). Dominant role of mitochondria in clearance of large Ca2+ loads from rat adrenal chromaffin cells. Neuron 16, 219-228. Hess, P. (1990). Calcium channels in vertebrate cells. Annual Review of Neuroscience 13, 337-356. Hirning, L.D., Fox, A.P., McCleskey, E.W., Oliver, B.M., Thayer, S.A., Miller, R.J. & Tsien, R.W. (1988). Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons. Science 239, 57-61. Holliday, J., Adams, R.J., Sejnowski, T.J. & Spitzer, N.C. (1991). Calcium-induced release of calcium regulates differentiation of cultured spinal neurons. Neuron 7, 787-796. Huettner, J.E. & Baughman, R.W. (1986). Primary cultures of identified neurons from the visual cortex of postnatal rats. Journal of Neuroscience 6, 3044-3060. Hodgkin A.L. & Keynes R.D. (1957). Movements of labeled calcium in squid giant axons. Journal of Physiology 134, 253-281. Holzapfel, S.W. (1968). The isolation and structure of cylopiazonic acid, a toxic metabolite of penicillium cylopium westling. Tetrahedron 24, 2101-2119. 110 Inesi, G. & Sagara, Y. (1994). Specific inhibitors of intracellular Ca2+-transport ATPases. The Journal of Membrane Biology 141, 1-6. Ingersoll, R.J. & Wasserman, R.H. (1971). Vitamin D3-induced calcium-binding protein binding characteristics, conformational effects, and other properties. Journal of Biological Chemistry 246, 2808-2814. Jensen, J.R. & Rehder, V. (1991). FCCP Releases Ca2+ from a non-mitochondrial store in an identified Helisoma neuron. Brain Research 551, 311-314. Kao, J.P.Y., Ffarootunian, A.T. & Tsien, R.Y. (1989). Photochemically generated cytosolic calcium pulses and their detection by fluo-3. Journal of Biological Chemistry 264, 8179-8184. Kao, J.P.Y. (1994). Practical aspects of measuring [Ca2+] with fluorescent indicators. Methods in Cell Biology 40, 155-181. Katz, S. & Blostein, R. (1975). Ca2+-stimulated membrane phosphorylation and ATPase activity of the human erythrocyte. Biochimica et Biophysica Acta 389, 314-324. Katz, B. & Miledi, R. (1969). Tetrodotoxin-resistant electric activity in presynaptic terminals. Journal of Physiology 203, 459-487. Kennedy, H.J. & Thomas, R.C. (1995). Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones. Journal of Physiology 484, 533-548. Kennedy, H.J. & Thomas, R.C. (1996). Effects of injection calcium-buffer solutions on [Ca2+]i in Voltage-clamped snail neurons. Biophysical Journal 70, 2120-2130. Kiedrowski, L. & Costa, E. (1995). Glutamate-induced destabilization of intracellular calcium concentration homeostasis in cultured cerebellar granule cells: role of mitochondria in calcium buffering. Molecular Pharmacology 47, 140-147. Ill Kiedrowski, L., Brooker, G., Costa, E. & Wroblewski, J.T. (1994). Glutamate impairs neuronal calcium extrusion while reducing the sodium gradient. Neuron 12, 295-300. Knauf, P. A., Proverbio, F. & Hoffmann, J.F. (1974). Electrophoretic separation of different phosphoproteins associated with Ca2+-ATPase and Na+, K+-ATPase in human red cells ghosts. Journal of General Physiology 63, 324-336. Koch, R.A. & Barish, M.E. (1994). Perturbation of intracellular calcium and hydrogen ion regulation in cultured mouse hippocampal neurons by reduction of the sodium ion concentration gradient. The Journal of Neuroscience 14, 2585-2593. Kostyuk, P.G., Mironov, S.L., Tepikin, A.L. & Belan, P.V. (1989). Cytoplasmic free Ca in isolated snail neurons as revealed by fluorescent probe Fura-2: Mechanisms of Ca recovery after Ca Load and Ca Release from intracellular stores. The Journal of Membrane Biology 110,11-18. Kraig, R., Petito, C, Plum, F. & Pulsinelli, W. Hydrogen ions kill brain at concentrations reached in ischemia. Journal of Cerebral Blood Flow & Metabolism 7, 379-386. Kretsinger, R.H., Nockolds, C.E. (1973). Carp muscle calcium-binding protein. II. Structure determination and general description. Journal of Biological Chemistry 248, 3313-3326. Lamb, G.D., & Stephenson, D.G. (1995). Activation of ryanodine receptors by flash photolysis of caged Ca2+. Biophysical Journal 68, 946-948. Lledo, P.M. Somasundaram, B., Morton, A.J., Emson, P.C., & Mason, W.T. (1992). Stable transfection of Calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis. Neuron 9, 943-954. Llinas, R., Sugimori, M., Lin, J.W. & Cherksey, B. (1989). Blocking and isolation of a 112 calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison. Proceedings of the National Academy of Sciences of the United States of America 86, 1689-1693. Luther, P.W., Yip, R.K., Bloch, R.J., Ambesi, A., Lindenmayer, G.E. & Blaustein, M.P. Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations. The Journal of Neuroscience 12, 4898-4904. Lynch, T.J. & Cheung, W.Y. (1979). Human erythrocyte (Ca2+ Mg2+)-ATPase: mechanism of stimulation by Ca2+. Archives of Biochemistry and Biophysics 194, 195-170. Lynch, G., Larson, J., Kelso, S., Barrionuevo, G. & Schottler, F. (1983). Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305, 719-21. MacLennan, D.H., Brandl, C.J., Korczak, B. & Green, N.M. (1985). Amino-acid sequence of a Ca2+-Mg2+ dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced • from its complementary DNA sequence. Nature 316, 696-700. Madison, D., Fox, A.P. & Tsien, R.W. (1987). Adenosine reduces an inactivating component of calcium current in hippocampal CA3 neurons. Biophysical Journal 51, 30a. Marengo, F.D., Wang, S.Y. & Langer, G.A. (1997). The effects of temperature upon calcium exchange in intact cultured cardiac myocytes. Cell Calcium 21, 263-273. Martinez-Zaguilan, R., Parnami, G. & Lynch, R.M. (1996). Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+. Cell Calcium 19, 337-349. Mayer, M.L., & Westbrook, G.L. (1987). Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurons. Journal of Physiology 394, 501 -527. McBurney R.N. & Neering I.R. (1987). Neuronal calcium homeostasis. Trends in 113 Neurosciences 10, 164-169. Meech, R.W. & Thomas, R.C. (1977). The effect of calcium injection on the intracellular sodium and pH of snail neurones. Journal of Physiology 265, 867-879. Meech, R.W. (1978). Calcium-dependent potassium activation in nervous tissues. Annual Review of Biophysics & Bioengineering 7, 1-18. Milanick, M.A. (1990). Proton fluxes associated with the Ca pump in human red-blood-cells. American Journal of Physiology 258, C552-C562. Miller, R.J. (1991). The control of neuronal Ca2+ homeostasis. Progress in Neurobiology 37, 255-285. Miller R.J. (1992). Voltage-sensitive Ca2+-channels. Journal of Biological Chemistry 267, 1403-1406. Minta, A., Kao, J.P.Y. & Tsien, R.Y. (1989). Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein. Journal of Biological Chemistry 264, 8171-8178. Mintz, I.M., Venema, VI, Swiderek, K.M., Lee, T.D., Bean, B.P. & Adams, M.E. (1992). P-type calcium channels blocked by the spider toxin omega-Aga-TVA. Nature 355, 827-829. Mironov, S.L. (1995). Plasmalemmal and intracellular Ca pumps as main determinants of slow Ca buffering in rat hippocampal neurones. Neuropharmacology 9, 1123-1132. Mironov, S.L., Usachev, Y.M. & Lux, H.D. (1993). Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons. Pflugers Archives 424, 183-191. Mitchell, P. (1966). Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biological Reviews of the Cambridge Philosophical Society 41, 445-502. 114 Moore, G.A., McConkey, DJ., Kass, G.E., O'Brien, P.J. & Orrenius, S. (1987). 2,5-di(tert-butyl)-l,4-benzohydroquinone-a novel inhibitor of liver microsomal Ca2+ sequestration. FEBSLetters 224, 331-336. Morgan, J.L, & Curran, T. (1988). Calcium as a modulator of the immediate-early gene cascade in neurons. Cell Calcium 9, 303-311. Morita, K., North, R.A. & Tokimasa, T. (1982). The calcium activated potassium conductance in guinea-Pig myenteric neurones. Journal of Physiology 329, 341-354. Nelson S.R. & Foltz F.M. (1983). Hypocalcemia and motor neuron degeneration in paraphysectomized frogs. Experimental Neurology 79, 763-772. Nerbonne, J.M. (1996). Caged Compounds: tools for illuminating neuronal responses and connections. Current Opinion in Neurobiology 6, 379-386. Nicholls, D.G. (1985). A role for the mitochondrion in the protection of cells against calcium overload? Progress in Brain Research 63, 97-106. Nicholls, D.G. (1986). Intracellular calcium homeostasis. British Medical Bulletin 42, 353-358. Niggli, V., Sigel, E. & Carafoli, E. (1982). The purified Ca2+ pump of human-erythrocyte membranes catalyzes an electroneutral Ca^-H* exchange in reconstituted liposomal systems. Journal of Biological Chemistry 257',2350-2356. Nowycky, M.C., Fox, A.P. & Tsien, R.W. (1985). Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440-443. Oberholtzer, J.C, Baettger, C, Summers, M.C. & Matschinsky, F.M. (1988). The 28 kDa calbindin is a major calcium binding protein in the basilar papilla of the chick. Proceedings of the National Academy of Sciences of the United States of America 85, 3387-3391. Osterrieder, W., Brum, G., Hescheler, J., Trautwein, W., Flockerzi, V. & Hofmann, F. (1982). Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current. Nature 298, 576-578. Ou Yang, Y., Kristian, T., Kristianova, V., Mellergard, P. & Siesjo, B.K. (1995). The influence of calcium transients on intracellular pH in cortical neurons in primary culture. Brain Research 676, 307-313. Park, Y.B., Herrington, J., Babcock, D.F. & Hille, B. (1996). Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells. Journal of Physiology 492, 329-346. Parsons, T.D., Ellis-Davies, G.C.R. & Aimers, W. (1996). Millisecond studies of calcium-dependent exocytosis in pituitary melanotrophs: comparison of the photolabile calcium-chelators nitrophenyl-EGTA and DM-nitrophen. Cell Calcium 19, 185-192. Plummer, M.R., Logothetis, D.E. & Hess, P. (1989). Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons. Neuron 2, 1453-1463. Putnam, R.W. (1995). Intracellular pH regulation. Cell Physiology Source Book 212-229. Randall, R.D. & Thayer, S.A. (1992). Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons. Journal of Neuroscience 12, 1882-1895. Rasmussen, U., Christensen, S.B. & Sanberg, F. (1978). Thapsigargin and thapsigargicine, two new histamine liberators from Thapsia garganica L. Acta Pharmaceutica Suecica 15, 133-140. 116 Reeves, J.P. & Sutko, J.L. (1980). Sodium-calcium exchange activity generates a current in cardiac membrane vesicles. Science 208, 1461-1464. Regan, L.J., Sah, D.Y.W., & Bean, B.P. (1991). Ca channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and co-conotoxin. Neuron 6, 269-280. Regehr, W.G. & Mintz, I.M. (1994). Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses. Neuron 12, 605-613. Reuter, H. & Porzig, H (1995). Localization and functional significance of the Na/Ca exchanger in presynaptic boutons of hippocampal cells in cultures. Neuron 15, 1077-1084. Reuter, H., Seitz, N. (1968). The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. Journal of Physiology 195, 451-470. Reuter, H., Stevens, C.F., Tsien, R.W. & Yellen, G. (1982). Properties of single calcium channels in cardiac cell culture. Nature 297, 501-504. Rizzuto, R., Simpson, A.W.M., Brini, M. & Pozzan, T. (1992). Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358, 325-327. Rizzuto, R., Bastianutto, S., Brini, M., Murgia, M. & Pozzan, T. (1994). Mitochondrial Ca2+ homeostasis in intact cells. The Journal of Cell Biology 126, 1183-1194. Ronner, P., Gazzotti, P. & Carafoli, E. (1977). A lipid requirement for the (Ca2+ Mg2+)-activated ATPase of erythrocyte membranes. Archives of Biochemistry and Biophysics 179, 578-583. Ross, W.N., Stockbridge, L.L. & Stockbridge, N.L. (1986). Regional properties of calcium 117 entry in barnacle neurons determined with Arsenazo HI and a photodiode array. Journal of Neuroscience 6, 1148-1159. Ross W.N. (1993) Calcium on the level. Biophysical Journal 64, 1655-1656. Sala, F. & Hernandez-Cruz, A. (1990). Calcium diffusion modeling in a spherical neuron: Relevance of buffering properties. Biophysical Journal 57, 313-324. Sanchez-Armass, S. & Blaustein, M.P. (1987). Role of sodium-calcium exchange in regulation of intracellular calcium in nerve terminals. American Journal of Physiology 252, C595-C603. Schatzmann, H.J. (1966) ATP-dependent Ca++ extrusion from human red cells. Experimentia 22, 364-368. Schlue, W.R. & Dorner, R. (1992). The regulation of pH in the central nervous system. Canadian Journal of Physiology and Pharmacology 70, S278-S285. Schwiening, C.J., Kennedy, H.J. & Thomas R.C. (1993) Calcium-hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons. Proceedings of the Royal Society London 253, 285-289. Schwiening, C.J. & Boron, W.F. (1994). Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na+-dependent Cl'-HCCVexchange. Journal of Physiology 475, 59-67. Seidler, N.W., Jona, I., Vegh, M. & Martonosi, A. (1989). Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264, 17816-17823. Shmigol, A, Kirischuk, S., Kostyuk, P. & Verkhratsky, A. (1994). Different properties of 11 caffeine-sensitive Ca2+ stores in peripheral and central mammalian neurones. Pflugers Archives 426, 174-176. Shuttleworth, T.J. & Thompson, J.L. (1991). Effect of Temperature on receptor-activated changes in [Ca2+]; and their determination using fluorescent probes. Journal of Biological Chemistry 266, 1410-1414. Sidky, AO. & Baimbridge, K.G. (1997). Na7Ca2+ exchange is not involved in the fast recovery of Ca2+ levels in neurons in which normal pHi is restored. Canadian Journal of Physiology and Pharmacology 75 Axvii. Siegl, P.K.S., Cragoe, E.J., Trumble, M.J. & Kaczorowski, G.J. (1984). Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride. Proceedings of the National Academy of Sciences of the United States of America 81, 3238-3242. Simpson, P.B., Challiss, R.AJ. 8c Nahorski, S.R. (1993). Involvement of intracellular stores in the Ca2+ responses to N-Methyl-D-aspartate and depolarization in cerebellar granule cells. Journal of Neurochemistry 61, 760-763. Simpson, P.B., Challiss R.A.J. & Nahorski, S.R. (1995). Neuronal Ca2+ stores: activation and function. Trends in Neurosciences 18, 299-306. Smallwood, J.L, Waisman, D.M., Lafreniere, D. & Rasmussen, H. (1983). Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange. Journal of Biological Chemistry 258, 1092-1097. Snutch, T.P., Leonard, J.P., Gilbert, M.M., Lester, H.A. & Davidson, N. (1990). Rat brain expresses a heterogeneous family of calcium channels. Proceedings of the National Academy of Sciences of the United States of America 87, 3391-3395. Stauffer, TP., Guerini, D., Celio, MR. & Carafoli, E. (1997). Immunolocalization of the 119 plasma membrane Ca2+ pump isoforms in the rat brain. Brain Research 748, 21-29. Stuenkel, E.L. (1994). Regulation of intracellular calcium and calcium buffering properties of rat isolated neurohypophysial nerve endings. Journal of Physiology 481, 251-271. Takadera, T., Shimada, Y. & Mohri, T. (1992). Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures. Brain Research 572, 126-131. Tang, CM., Dichter, M. & Morad, M. (1990). Modulation of the N-methyl-D-aspartate channel by extracellular EE. Proceedings of the National Academy of Sciences of the United States of America 87, 6445-6449. Tatsumi, F£. & Katayama, Y. (1993). Regulation of the intracellular free calcium concentration in acutely dissociated neurones from rat nucleus basalis. Journal of Physiology 464, 165-181. Thastrup, O., Dawson, A.P., Scharff, O., Foder, B., Cullen, P.J., Drobak, B.K., Bjerrum, P.J., Christensen, S.B. & Hanley, M.R. (1989). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents & Actions 27, 17-23. Thayer, S.A. & Miller, R.J. (1990). Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro. Journal of Physiology 425, 85-115. Traynelis, S.F. & Cull-Candy, S.G. (1990). Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons. Nature 345, 347-350. Tsien, R.W., Lipsombe, D., Madison, D.V., Bley, KR. & Fox, AP. (1988). Multiple types of neuronal calcium channels and their selective modulation. Trends in Neurosciences 11,431-438. Tsien, R.W. & Tsien, R.Y. (1990). Calcium channel stores and oscillations. Annual review of 120 Cell Biology 6, 715-760. Tsien, R.W. Ellinor, P.T. & Home, W.A. (1991). Molecular diversity of voltage dependent Ca2+ channels. Trends in Neurosciences 12, 349-354. Tymianski, M. Charlton, M.P., Carlen, P.L. & Tator, CH. (1993). Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. The Journal of Neuroscience 13, 2085-2104. Vasington, F.D., Murphy, J. (1961). Federation Proceedings 20, 146. Wang, G.J., Randall, R.D. & Thayer, S.A. (1994). Glutamate induced intracellular acidification of cultured hippocampal neurons demonstrates altered energy metabolism resulting from Ca2+ loads. Journal of Neurophysiology 72, 2563-2569. Wasserman, R.H. & Taylor, A.N. (1966). Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science 152, 791-793. Werth, J.L. & Thayer, S.A. (1994). Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons. The Journal of Neuroscience 14, 348-356. Werth, J.L., Usachev, Y.M. & Thayer, S.A. (1996). Modulation of calcium efflux from cultured rat dorsal root ganglion neurons. The Journal of Neuroscience 16, 1008-1015. White, R.J. & Reynolds, I.J. (1995). Mitochondria and Na7Ca2+ exchange buffer glutamate-induced calcium loads in cultured cortical neurons. The Journal of Neuroscience 15, 1318-1328. Widdowson, E.M. & Dickerson, J.W. (1964). Mineral Metabolism 2, 1-247. 121 Zavoico, G.B. & Cragoe, E.J. (1988). Ca2+ mobilization can occur independent of acceleration of Na+/F£+ exchange in thrombin-stimulated human platelets. Journal of Biological f Chemistry 263, 9635-9639. Zucker, R.S. (1992). Effects of photolabile calcium chelators on fluorescent calcium indicators. Cell Calcium 13, 29-40. 

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
France 6 0
United States 4 3
China 4 0
Taiwan 4 0
Canada 2 0
Australia 1 0
Italy 1 0
City Views Downloads
Unknown 14 3
Beijing 2 0
Ashburn 2 0
Wilmington 1 0
Bologna 1 0
Shenzhen 1 0
Shanghai 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0087958/manifest

Comment

Related Items