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The effects of norepinephrine on the activity of the Na+/H+ exchanger in acutely dissociated adult rat… Smith, Garth Andrew Michael 1996

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THE EFFECTS OF NOREPINEPHRINE ON THE ACTIVITY OF THE Na/H 4 EXCHANGER IN ACUTELY DISSOCIATED ADULT RAT HIPPOCAMPAL CA1NEURONS by G A R T H A N D R E W M I C H A E L SMITH B.Sc. (Neuroscience), The University of Toronto, 1990 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Physiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A October, 1996 © Garth Andrew Michael Smith, 1996 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 ^ t ^ ^ i & l o The University of British Columbia Vancouver, Canada Date K V J H\<\(J> • DE-6 (2/88) 11 ABSTRACT The effects of norepinephrine on resting intracellular pH (pH;) and on the rate of recovery of pH; following an imposed intracellular acid load were investigated in acutely dissociated adult rat hippocampal CA1 neurons loaded with the fluorescent hydrogen ion indicator, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. During perfusion with HC0 37C0 2-free, HEPES-buffered media, the dominant acid extrusion mechanism utilized by these neurons was an amiloride-insensitive variant of the Na7H + exchanger. In HC037C02-buffered saline, acid extrusion was supplemented by the activity of a 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid-sensitive, Na + -dependent HCOy/Q" exchanger which also contributed to the maintenance of resting pH :. Norepinephrine 10 u M evoked a rise in resting pH ; that was long-lasting and increased the rate of pH ; recovery from an imposed intracellular acidification in both the absence and presence of external HCGy. The effects of norepinephrine were attenuated by the non-selective R-adrenergic receptor antagonist, propranolol, and were mimicked both by the mixed (3-adrenergic receptor agonist isoproterenol and by the selective p,- and p2-adrenergic receptor agonists dobutamine and terbutaline, respectively, cc-adrenoceptor agonists affected neither steady-state pLf nor the rate of pHj recovery from an imposed acid load. The norepinephrine-induced increase in resting pH; and in the rate of pH; recovery from an imposed acid load were independent of changes in intracellular free [Ca2+] and were mimicked by the acute application of cholera toxin, forskolin or Sp-cAMPS in the absence of norepinephrine. Conversely, the effects of norepinephrine were occluded by 2',5'-dideoxyadenosine, Rp-cAMPS and H-89. I l l The results indicate not only that norepinephrine can augment the activity of the Na7H + exchanger through the activation of p-adrenergic receptors, G s a , cAMP, and protein kinase A but also that the second messengers themselves are able to alter the activity of the Na7H + exchanger. The ability of norepinephrine to alter pH, via modulation of the activity of the dominant acid extrusion mechanism present in rat hippocampal C A 1 neurons may constitute a mechanism to dissipate rapidly intracellular acid loads produced by the actions of other neurotransmitters such as glutamate and may subserve some of the reported P-adrenergic receptor-mediated effects of norepinephrine in the C A 1 region of the hippocampus. I V TABLE OF CONTENTS Page Abstract .' i i Table of Contents iv List of Tables vi List of Figures vii Acknowledgments xi INTRODUCTION 1 Extracellular pH and neuronal activity 2 Modulation of pHj by cellular events 6 pHj modulation of cellular events 7 Characteristics of Na7H + and HC037C1" exchangers 11 Modulation of pH, regulating mechanisms by extracellular agents and second messengers 14 Norepinephrine and the hippocampus 19 Overview 21 M A T E R I A L S A N D METHODS 23 Cultured post-natal rat hippocampal neurons 23 Acutely dissociated adult rat hippocampal CA1 neurons 24 Cell loading and BCECF 26 Experimental setup and the ratiometric method 28 Solutions and chemicals 30 Experimental paradigms 32 Calculation of pH; and analysis of data 34 RESULTS . 46 Characterization of acid extrusion mechanisms 46 Acid extrusion during perfusion with HC03"-free, HEPES-buffered medium 47 Mechanisms of acid extrusion in the presence of HC0 3 " 48 Effects of norepinephrine on pH> 50 Adrenergic receptor subtypes mediating the effects of norepinephrine on the activity of the Na7H + exchanger .: 53 Intracellular mechanisms of norepinephrine-induced actions on pH; 56 V Page Involvement of the Gs-isoform of guanine nucleotide-binding regulatory proteins 56 Involvement of second messengers: c A M P and calcium 59 Involvement of protein kinase A 63 DISCUSSION .' 135 Acid extrusion mechanisms present in adult rat hippocampal CA1 neurons 135 Effects of norepinephrine on pH, 137 Effects of norepinephrine on mammalian central neuronal excitability 140 Signal transduction systems and pHj 143 Effects of activation of adenylyl cyclase and protein kinase A on neuronal excitability 147 Physiological significance of norepinephrine- and second messenger-induced changes in pH ; 148 REFERENCES 154 vi LIST OF TABLES Page Table 1 Composition of HEPES-buffered experimental solutions 40 Table 2 Composition of HCO37C02-buffered experimental solutions 41 vii LIST OF FIGURES Page Figure 1 Sample calibration plot for BCECF 42 Figure 2 Control acid load recoveries during perfusion with HEPES-buffered medium 44 Figure 3 Distributions of steady-state pHj values for all neurons during perfusion with HEPES-buffered and HC037C02-buffered media 65 Figure 4 Effects of EIPA on steady state pHj and on the recovery of pH ; from an intracellular acid load in HEPES-buffered media 67 Figure 5 Effect on steady-state pHj of perfusion with Na+-fee, HEPES-buffered media 69 Figure 6 Effects of perfusion with Na+-free, HEPES-buffered media on recovery of pHj from an imposed intracellular acidification 71 Figure 7 The effects of the transition from HEPES-buffered saline to HCOy/COj-buffered medium on resting pH ; 73 Figure 8 Effects of DIDS and 0 [Cl"] 0 on steady-state pH t in the presence of H C O 3 " 75 Figure 9 Effects of 0 [Na + ] 0 on resting pHj and on the recovery of pH; from an imposed intracellular acidification in the presence of H C O y 77 Figure 10 Effects of norepinephrine on steady state pH; and on the rate of pH; recovery from an imposed cytosolic acidification, in the presence o f H C 0 3 - a t 3 7 ° C 79 Figure 11 Effects of norepinephrine on steady state pH ; and on the rates of pH, recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium 81 Figure 12 Long-term effect of norepinephrine on steady-state pHj during perfusion with HEPES-buffered medium 83 Figure 13 Comparison of the control rates of pH; recovery to rates of pH ( recovery under the influence of norepinephrine during perfusion with HEPES-buffered medium 85 V l l l Page Figure 14 Effect of norepinephrine on steady state pH ; during perfusion with a Na+-free, HEPES-buffered medium 87 Figure 15 Effects of 6-fluoronorepinephrine and isoproterenol on steady state pHj during perfusion with HEPES-buffered medium 89 Figure 16 Effects of 6-fluoronorepinephrine and isoproterenol on rates of pH ( recovery from an imposed intracellular acidification in HEPES-buffered medium 91 Figure 17 Comparison of the rates of pH; recovery under the influence of either isoproterenol or 6-fluoronorepinephrine and control rates of pH; recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium 93 Figure 18 Effects of dobutamine on steady state pH; and on the rate of pH ; recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium 95 Figure 19 Concentration dependent effects of terbutaline on steady state pH, during perfusion with HEPES-buffered saline 97 Figure 20 Effect of terbutaline on the rates of recovery of pH ; from an imposed intracellular acid load during perfusion with HEPES-buffered medium 99 Figure 21 Effect of norepinephrine and p-adrenergic receptor agonists on the distribution of the steady state pH : values of adult rat hippocampal CA1 neurons during perfusion with HEPES-buffered media 101 Figure 22 Effects of 10 uM norepinephrine with 20 u M propranolol on steady state pHj and on the rate of pH ; recovery after an imposed intracellular acid load during perfusion with HEPES-buffered medium 103 Figure 23 Comparison between control rates of pH, recovery and rates of pH, recovery under the influence of 10 uM norepinephrine with 20 u M propranolol during perfusion with HEPES-buffered medium 105 Figure 24 Effect of a pre-treatment with cholera toxin on the norepinephrine-induced increase in steady state pH; during perfusion with HEPES-buffered medium 107 ix Page Figure 25 Effect of a pre-treatment with cholera toxin on the norepinephrine-induced increase in the rate of pH, recovery from an imposed acid load during perfusion with HEPES-buffered medium 109 Figure 26 Effects of an acute exposure to cholera toxin on resting pH; and a comparison of the rates of pH; recovery under the influence of cholera toxin and norepinephrine in cultured post-natal hippocampal neurons during perfusion with HEPES-buffered medium I l l Figure 27 Effects of cholera toxin on steady state pHj and on the recovery of pH; from an imposed intracellular acidification in freshly isolated adult hippocampal CA1 neurons perfused with HEPES-buffered medium 113 Figure 28 Effects of forskolin on steady state pH : and on the rates of pH s recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium 115 Figure 29 Effects of 2',5'-dideoxyadenosine on norepinephrine-induced effects on steady state pH ( and on the rates of pHi recovery from an imposed acid load during perfusion with HEPES-buffered medium 117 Figure 30 Effects of l',9'-dideoxyforskolin on steady state pHj and on the rates of pH ; recovery from an imposed acid load during perfusion with HEPES-buffered medium 119 Figure 31 Effect of I B M X on steady state pH> during perfusion with HEPES-buffered medium 121 Figure 32 Effects of 0 [Ca2 +]0 on norepinephrine-induced changes in steady state pHj and in the rates of pH : recovery from an imposed acid load during perfusion with HEPES-buffered medium 123 2+ Figure 33 Effects of perfusion with a Ca -free, HEPES-buffered medium on isoproterenol-induced changes in steady state pHj and in the rates of pHj recovery from an imposed acid load 125 Figure 34 Effect of isoproterenol on steady state intracellular Ca 2 + during perfusion with Ca2+-free, HEPES-buffered medium 127 Figure 35 Effects of Sp-cAMPS on steady state pHj and on the rates of pH, recovery from an imposed acid load during perfusion with HEPES-buffered medium 129 X Page Figure 36 Effects of Rp-cAMPS on norepinephrine-induced actions on steady state pH ; and on the rates of pHj recovery from an imposed acid load during perfusion with HEPES-buffered medium 131 Figure 37 Effects of H-89 on norepinephrine-induced effects on steady state pH ; and on the rates of pH ; recovery from an imposed acid load during perfusion with HEPES-buffered medium 133 ACKNOWLEDGMENTS First and foremost, I would like to thank sincerely Dr. John Church for his support and patience during the past two years. After observing his aptitude as a researcher and his pursuit of excellence, my future endeavors will definitely be influenced. A debt of gratitude is owed to the members of my supervisory and examining committees: Drs. Kenneth Baimbridge, Vladimir Palaty, Timothy Murphy, Raymond Pederson, Lynn Raymond and Steven Kehl. Their encouragement and helpful discussion aided greatly in the completion of my thesis. I would also like to thank colleagues and faculty members of the Departments of Physiology and Anatomy for providing an excellent environment for both intellectual and personal growth. Special thanks must go to Stella Amatjda and Joe Tay for their technical expertise. I am also greatly indebted to my friend, Keith Baxter whose previous work facilitated greatly the progress of my research. Deepest regards to Shahnawaz Virani and my labmate, Claire Thurgur, for their honesty, caring and generosity. Finally, I am grateful to my family, especially Danielle and Rebecca for their unconditional love. 1 Introduction The evolution of intracellular pH (pHi) regulatory mechanisms resulted from the need to regulate strictly cytoplasmic levels of the highly reactive H + ion (Thomas, 1984). Internal production of protons via metabolism and the inward electrochemical gradient for H + , due to the membrane potential, represent a constant source of protons that need to be actively extruded (Busa and Nuccitelli, 1984). This active regulation of pHj depends on a variety of transport mechanisms, such as the N a + / H + exchanger and the N a + -dependent H C C Y / C I - exchanger, that extrude H + or introduce bicarbonate ions respectively, to alleviate a decrease in pHj (Roos and Boron, 1981; Thomas, 1984). Conversely, following an intracellular alkaline load, mechanisms such as the Na + -independent (i.e. passive) H C O 3 V C I " exchanger will extrude bicarbonate ions to restore "normal" pH; (Roos and Boron, 1981; Thomas, 1984). In the mammalian nervous system, several factors can influence pH ; , including extracellular pH (pH 0), normal neuronal activity and pathophysiological events such as seizure activity and ischemia/hypoxia (Chesler, 1990; Chesler and Kaila, 1992). In turn, alterations in pH s have been suggested not only to act as a signal necessary for normal cellular processes but also to mediate a diverse array of neurophysiological effects, such as changes in neuronal excitability. However, in peripheral cell types, it is also established that pH, can be modulated by external agents such as hormones, growth factors and neurotransmitters via surface receptor-mediated pathways which act to alter 2 the activity of pHt regulating mechanisms (Moolenaar et al, 1983; Wakabayashi et al, 1992). The present study investigates the possibility that external agents are able, in a similar fashion, to modulate the activities of acid extrusion mechanisms in mammalian central neurons. Extracellular pH and neuronal activity The excitability of mammalian neurons can be modulated by physiologically relevant changes in pH 0 (Aram and Lodge, 1987; Balestrino and Somjen, 1988; Church and McLennan, 1989; Gottfried and Chesler, 1994; Tombaugh and Somjen, 1996). In acutely dissociated adult rat hippocampal CA1 pyramidal neurons, the activation and inactivation properties of voltage-gated sodium, calcium and potassium channels can be altered by relatively modest shifts in external pH and thereby affect the excitability of these neurons (Tombaugh and Somjen, 1996). Another example of such an interaction involves the well-documented proton sensitivity of the JV-methyl-D-aspartate (NMDA) subtype of glutamate receptor. Increases or decreases in pH 0 will respectively augment and reduce the responsiveness of hippocampal neurons to excitatory amino acids such as N M D A . Thus, increased levels of extracellular protons (i. e. decreases in pH 0) can inhibit current flow through the N M D A receptor-operated channel with a 50% inhibitory concentration (IC 5 0) of ~ pH 6.9 - 7.3 (e.g. Tang et al., 1990; Traynelis and Cull-Candy, 1990; Vyklicky et al., 1990). The inhibitory effect of extracellular protons appears to involve a decrease in the opening frequency of the receptor-channel complex and is controlled by an aspartate residue in a putative extracellular loop of the N M D A receptor-channel complex (Vyklicky et al, 1990; Traynelis et al, 1995; Kashiwagi et al, 1996). Lowering p H 0 completely and reversibly suppresses low-Mg -induced, N M D A receptor-mediated epileptiform bursting in neocortical slices (Aram and Lodge, 1987) and combined entorhinal cortex-hippocampal slices (Velisek et al, 1994) whereas raising p H 0 evokes epileptiform activity in hippocampal slices which is sensitive to N M D A receptor antagonists (Church and McLennan, 1989). Not only can changes in pH 0 affect ligand-gated and voltage-operated ion channels, but neuronal activity itself can lead to alterations in p H 0 (reviewed by Chesler and Kaila, 1992). For example, in the CA3 region of guinea-pig hippocampal slices, repetitive electrical stimulation resulted in a transient increase in p H 0 (~ 0.2 pH units) followed by a more prolonged decrease in pH 0 (~ 0.1 pH units) that lasted 1-4 minutes (Jarolimek et al, 1989). Similarly, in guinea-pig and rat hippocampal slices, application of glutamate or y-aminobutyric acid (GABA) evokes a transient extracellular alkalosis followed by a smaller acid transient (Jarolimek et al, 1989; Chen and Chesler, 1992a). The mechanism(s) underlying glutamate-induced increases in p H 0 appear distinct from those controlling the extracellular alkalosis evoked by G A B A (Jarolimek et al, 1989; Chen and Chesler, 1992a; Paalasmaa and Kaila, 1996). GABA-induced increases in p H 0 are mediated by an efflux of bicarbonate ions through G A B A A receptor-operated anion channels (Kaila and Voipio, 1987; Bormann et al, 1987; Chen and Chesler, 1992a; Kaila et al, 1993). In contrast, both N M D A and non-NMDA subtypes of glutamate receptors are implicated in stimulus-evoked, glutamate-mediated increases in pH 0 (Chen and Chesler, 1992b; Voipio et al, 1995). Both N M D A - and non-NMDA-induced changes in 4 p H 0 are not due a ligand-activated proton conductance but are linked to an influx of external calcium through both NMDA-operated and voltage-gated C a 2 + channels (Paalasmaa et al, 1994; Smith et al, 1994; Paalasmaa and Kaila, 1996). The resultant rise in intracellular [Ca 2 +] may then activate C a 2 + / H + exchange (Ca 2 + -H + ATPase) which 2+ will lead to a decrease in both [Ca ] ; and pH s and an extracellular alkaline transient (Schwiening et al, 1993). It has been demonstrated that the transient extracellular alkalosis induced by excitatory synaptic transmission can, in turn, modulate the activity of the N M D A receptor-channel complex within a physiologically relevant time frame (Taira et al, 1993; Gottfried and Chesler, 1994). Therefore, it is apparent that pH 0 can both influence and be influenced by neuronal activity. The changes in pH 0 resulting from normal neuronal activity are quite modest. However, certain pathophysiological events such as cerebral ischemia and epileptic seizures, can produce not only a significant increase in intracellular [Ca2 +] but can also be accompanied by a reduction in tissue pH which, depending on its magnitude, may mediate subsequent neuronal damage or act in a neuroprotective manner (von Hanwehr et al, 1986; Siesjo et al, 1985; Choi, 1988). During ischemia, the increased amount of extracellular protons and acid equivalents originates from the increased operation of anaerobic glycolysis and the subsequent production of lactic and carbonic acids (Siesjo, 1985). In rat hippocampal slices, oxygen-glucose deprivation-induced increases in intracellular [Ca ] (mediated by the influx of Ca ions) can be blocked by mild extracellular acidosis (~ pH 6.8) and enhanced by an increase in pH 0 (~ pH 7.8; Ebine et al, 1994). External acidosis, if not too severe, may be neuroprotective possibly by 5 reducing Ca -influx via N M D A receptor-operated channels and voltage-gated calcium channels or by attenuating Na + entry through voltage-gated Na + channels (Andreeva et al., 1992; Kaku et al., 1993; Simon et al, 1993; Kristian et al, 1994; Tombaugh, 1994). Clinically, hyperventilation-induced extracellular alkalosis can evoke seizures in epileptic patients while the resultant tissue acidosis caused by epileptic seizures may feed back to reduce the duration of the seizures (Somjen, 1984; Velisek et al, 1994). In contrast, Kraig et al. (1987) injected sodium lactate into rat parietal cortices to reproduce p H 0 levels which can be observed during complete cerebral ischemia, i.e. below pH 5.3, and this resulted in local areas of tissue necrosis reminiscent of those observed after ischemic brain infarction. Similar results have been obtained in cultured rat forebrain neurons and in hippocampal slices (Goldman et al, 1989; Morimoto et al, 1994). Therefore, it is apparent that modest reductions in pH 0 may be neuroprotective and also exert antiepileptiform/anticonvulsant activity. However, it is also apparent that marked reductions in p H 0 may be detrimental to neuronal survival, possibly due to a concomitant decrease in pHj (Nedergaard et al, 1991; see below). Furthermore, it is possible that some of the effects of even mild changes in pH 0 , noted above, may reflect alterations in pHj consequent upon changes in pH 0 . Previously, it was thought that changes in p H 0 did not affect neuronal pHj. However, it is now established that any change in p H 0 will result in a slightly smaller shift in pHj in the same direction (Church and Baimbridge, 1991; Nedergaard et al, 1991; Ou-yang et al, 1993). Therefore some of the noted effects of pH 0 on both the normal activity of neurons and on pathophysiological events may be mediated by alterations of pHj. 6 Modulation of pH-t by cellular events Neuronal depolarization will , in most cases, produce a decrease in pHj (Chesler, 1990, but see Ou-yang et al., 1995). This activity-induced intracellular acid shift is mediated by increased levels of metabolic end-products such as carbon dioxide and lactic acid (Siesjo, 1985), a rise in intracellular calcium which leads to the release of protons from internal stores (Chesler, 1990) and activation of Ca -H exchange (Schwiening et al, 1993), and/or by an efflux of bicarbonate ions via voltage- or ligand-gated channels (Chen and Chesler, 1992a; Kaila etal, 1993). In cultured hippocampal neurons, 10 uM glutamate evoked an average decrease in pHj of 0.30 - 0.50 pH units (Hartley and Dubinsky, 1993; Irwin et al., 1994; Wang et al, 1994). This effect was N M D A receptor-mediated and was dependent on a rise in 2+ intracellular [Ca ] (see above) but was abolished when glucose was substituted by 2-deoxyglucose, implicating an increased production of lactic and carbonic acids as the cause of the drop in pH; (Wang et al, 1994; Irwin, 1994). In motor neurons of the frog spinal cord, 30 u M N M D A and/or repetitive stimulation of the dorsal root produced an average intracellular acidification of 0.21 pH units (Endres et al., 1986). This response of pH; to activation of the N M D A receptor-channel complex has also been seen in catfish retinal horizontal cells and neocortical neurons (Dixon, 1993; Ou-yang et al., 1995). Amos and Richards (1994) also proposed a role for metabotropic glutamate receptors in the glutamate-induced decrease in pH ; in cultured neocortical neurons, suggesting that the G-protein-linked receptor may act on pHj regulating mechanisms to initiate the change in p H , 7 Therefore, it is apparent that neuronal activity can lead to marked changes in pHj. These activity-induced changes in pHj may in turn act to modulate a number of cellular processes. pHt modulation of cellular events Many biologically active proteins such as enzymes, ion transporters and ion channels are known to be sensitive to the intracellular H + concentration (Siesjo, 1985; Boron, 1989). Presumably, because the charge(s) on ionizable groups in proteins can be affected by changes in pH i 3 the structural configuration of proteins and, thus, their activity could be altered (Siesjo, 1985). In rat hippocampal neurons, for example, the plasmalemmal C a 2 + pump can be suppressed by intracellular acidification (Carafoli, + 2"F 1987). And in many cell types, the activity of the Na -Ca exchanger is dependent upon pHj (e.g. Doering and Lederer, 1993; Koch and Barish, 1994). Significant changes in cellular activity can also be attributed to small perturbations in pHj (Chesler, 1990). In several invertebrate preparations, changes in pH ; can influence N a + , K + , CI" and C a 2 + currents (Baker and Honerjager, 1978; Wanke et al, 1979; Carbone et al, 1981; Umbach, 1982; Moody, 1984). For example, both the inwardly rectifying and delayed rectifier K + conductances in squid axons and Helix aspersa neurons are blocked by a small decrease in cytosolic pH (Wanke et al, 1979; Meech, 1979; Moody, 1984). And in 2+ leech Retzius neurons, the open probability of both the ATP-inhibited and Ca -dependent K + channels are sensitive to alterations in pHj (Frey et al, 1993). 8 Many studies have indicated that a variety of ionic conductances in vertebrate central neurons and peripheral cell types are also sensitive to changes in cytosolic pH. For example, Daumas and Andersen (1993) found that intracellular acidification blocked rat brain N a + channels incorporated into lipid bilayers and, in cultured rat hippocampal neurons, Mironov (1995) reported an increase and a decrease in high-[K+]0-evoked 2_j  [Ca ]j transients during intracellular alkalosis and acidosis, respectively. Similarly, potentials mediated by Ca -activated K conductances in rat hippocampal CA1 pyramidal neurons (Church, 1992) and voltage-gated K + channels in rat dorsal vagal motoneurons (Cowan and Martin, 1996) also appear sensitive to changes in pHj. Mironov and Lux (1991) reported that cytoplasmic alkalinization increases high-2+ threshold Ca currents in chick dorsal root ganglion neurons, while in horizontal cells of fish retina a decrease in pHj leads to a reduction in the high voltage-activated, nifedipine-2"T~ 2+ and Cd -sensitive Ca current (Takahashi et al., 1993). Also, in guinea-pig ventricular 2+ myocytes, L-type Ca channels are sensitive to changes in pH; (Irisawa and Sato, 1986; Kaibara and Kameyama, 1988; Prod'hom et al, 1987; Pietrobon et al, 1989; Takahashi 2+ + et al, 1993). Finally, Ca -activated K conductances decrease upon intracellular acidification in rat skeletal muscle (Laurido et al, 1991), in rabbit tracheal smooth muscle (Kume et al, 1990) and in rat carotid body cells (Peers and Green, 1991). In addition to effects on ionic conductances, changes in pHj can also affect other cellular functions. Electrophysiological and Lucifer yellow dye-coupling studies have determined that gap junctional conductance increases with a decrease in the concentration of intracellular protons, and vice versa, in guinea-pig and rat hippocampal neurons 9 (MacVicar and Jahnsen, 1985; Spray and Bennett, 1985; Church and Baimbridge, 1991). There is strong evidence for the involvement of changes in the activity of pHj regulating mechanisms, and thus pH i ; in cellular processes such as the onset and/or the maintenance of cellular proliferation (Moolenaar, 1986; Busa, 1986; Grinstein et al, 1989). In rat spinal cord astrocytes, for example, the mitotic rate is highly dependent on pH; where the highest rate of proliferation occurs at ~ pHj 6.7 (Pappas et al, 1994). Also, mutant Chinese hamster fibroblasts, devoid of pH; regulating mechanisms, failed to proliferate or reinitiate D N A synthesis in a pH 0 of less than 7.2; an increase of pH s by 0.2 pH units, at a constant pH 0 , restored full mitogenic response (Wakabayashi et al, 1992). Interestingly, virtually all agents that have mitogenic potential such as growth factors, chemostatic peptides, mitogenic lectins and neurotransmitters can also interact with one or more acid extrusion mechanism(s), including the ubiquitous N a + / H + exchanger-1 isoform (NHE-1), via a variety of second messenger systems (Moolenaar et al, 1983; Moolenaar, 1986; Frelin et al, 1988; Grinstein et al, 1989; Wakabayashi et al, 1992; see below). A l l of these mitogens modulate the activity of the acid extrusion mechanisms by increasing their affinity for intracellular protons (Grinstein et al, 1989; Wakabayashi et al, 1992). Metabolism can also be regulated by shifts in pH, because the active sites on certain crucial enzymes often contain ionizable groups whose state of ionization may affect the enzyme's conformation and thus its substrate-binding and catalytic properties (Roos and Boron, 1981, Bazaes and Kemp, 1990). Therefore fluctuations of pHj can influence many aspects of cellular function from responses to external signals to initiation of growth and development of the cell. 10 As noted above, cerebral ischemia is accompanied not only by extracellular acidosis but also by a decrease in cytosolic pH (Siesjo, 1985). Nedergaard et al. (1991) suggested that the duration and degree of exposure to an acidified cytoplasm were the determining factors of acid-induced neuronal death. In this study, two patterns of acid-induced cell death emerged. Moderate levels of intracellular acidification initiated the degradation of the neuron only after a long latency period (~ 24 - 48 hours) while excessive increases of internal protons rapidly caused cell mortality (Nedergaard et al., 1991; see also Shen et al, 1995). Possible mechanisms mediating the effects of marked decreases in pHi on neuronal survival include the slow depletion of energy stores and/or an associated denaturation of integral proteins (Nedergaard et al, 1991). The lowering of pHj to levels encountered during cerebral ischemia can also lead to a markedly enhanced initiation and propagation of free radical reactions which also may form the basis of the detrimental actions of excessive intracellular acidosis (Siesjo, 1985). In summary, changes in pHj are associated with significant effects on neuronal function, both during normal activity and during neural pathologies. It is clear that neurons must not only be able to maintain strict control of their resting pH i 5 but must also be able to recover from alterations in their cytosolic pH. Neurons will utilize pHi regulatory systems such as the N a + / H + exchanger and HCGy/Cl" exchangers to perform these tasks. 11 Characteristics ofNa+/H+ and HCOf/Ct exchangers The electroneutral N a + / H + exchanger extrudes protons at the expense of the inward electrochemical gradient for N a + across the plasma membrane (Roos and Boron, 1981; Thomas, 1984). The exchanger is activated by a decrease in pHj and its activity is dependent on both extracellular [Na+] and pH 0 , i.e. a fall in pH 0 inhibiting it (Aronson, 1985; Grinstein and Rothstein, 1986). Five isoforms of the N a + / H + exchanger have to date been cloned, N H E 1-4 from mammalian species and pNHE from trout erythrocytes (Orlowski et al, 1992; Borgese et al, 1994; Yun et al, 1995). N H E 1 - 4 are present to varying degrees in the plasma membranes of most mammalian cells, although a brain-specific isoform has not yet been described (Clark and Limbird, 1991). The different isoforms of the exchanger display a varied sensitivity to the diuretic amiloride and its 5-N substituted derivatives such as 5-(Af-ethyl,./V-isopropyi)-amiloride (EIPA) and 5-(N-methyl-A^-guanidinocarbonylmethyl) amiloride (MGCMA), which compete with sodium for the external transport site (Clark and Limbird, 1991). A l l of the cloned isoforms of the N a + / H + exchanger possess two separate functional domains: a N-terminal hydrophobic domain spanning the plasma membrane 10-12 times which regulates ion transport and a second hydrophilic portion of the protein facing the cytoplasm which is crucial in determining the "set-point" value of the H + sensor system (Orlowski et al, 1992; Yun et al, 1995). The cytoplasmic tail of the membrane protein is required for the modulation of the exchanger by receptor-mediated pathways due to the presence of possible consensus sites for phosphorylation by various protein kinases (Grinstein and 12 Rothstein, 1986; Winkel et al, 1993; Borgese et al, 1994, Yun et al, 1995). The integral plasma membrane protein has another proton binding site termed the proton sensor or regulator in addition to the proton transport site on the intracellular face of the exchanger. The proton sensor site must be occupied for the protein to undergo the conformational change necessary for the exchanger to transport intracellular protons (Aronson, 1985; Wakabayashi etal, 1992). The gene family that encodes the plasma membrane H C O 3 V C I " exchangers contains three members, A E 1-3 (Kopito, 1990). The isoform-specific functional differences are not clearly defined, although they do display a characteristic tissue distribution (Kopito et al, 1989; Kopito, 1990; Humphreys et al, 1995). Bicarbonate-transport systems can act either as acid or base extruders depending on the relative gradients for both H C O 3 " and CI" ions across the plasma membrane and their dependence on extracellular N a + (Frelin et al, 1988). The Na+-dependent HC0 3 7C1" exchanger extrudes acid equivalents (by importing H C O 3 " ions) under normal physiological conditions whereas the Na+-independent form acts primarily to extrude H C 0 3 " ions and is thus an acid loader. Both are inhibited by stilbene derivatives such as 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS). The first report characterizing pH, regulating mechanisms in vertebrate neurons was from Chesler (1986) who found that lamprey reticulospinal neurons possessed a N a + / H + exchanger and a HC03"-dependent acid extrusion mechanism. The major pH t regulating mechanisms operating in several types of mammalian central neurons have 13 now been characterized. In fetal, neonatal and adult rat hippocampal neurons, both the Na+-dependent and Na+-independent HC0 3"/C1" exchangers and an amilonde-insensitive N a + / H + exchanger have been identified (Raley-Susman et al., 1991 & 1993; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996). The N a + / H + exchanger appears to be the primary acid extrusion mechanism in cortical neurons and fetal and mature (21- to 30-day old) hippocampal CA1 neurons (Raley-Susman et al., 1991; Ou-yang et al., 1993; Baxter and Church, 1996; Bevensee et al., 1996). In contrast, Schwiening and Boron (1994) proposed that a Na+-dependent HC0 37Cr exchanger was the dominant acid extrusion mechanism in neonatal hippocampal CA1 neurons. The presence of HC0 3 7C1" and N a + / H + exchangers has also been determined in rat brain synaptosomes (Nachshen and Drapeau, 1988; Sanchez-Armass et al., 1994; Martinez-Zaguilan et al., 1994), cultured cerebellar Purkinje cells (Gaillard and Dupont, 1990) and cultured sympathetic neurons (Tolkovsky and Richards, 1987). Baxter and Church (1996) determined that, in cultured fetal hippocampal neurons, the activities of the N a + / H + and Na+-dependent, HCO3VCI" exchangers were temperature-dependent. At room temperature, both exchangers participated in the maintenance of steady state pHj but, at 37°C, N a + / H + exchange dominated acid extrusion. Their results also suggested the presence of a Na+-independent, HC0 37Cr exchanger that, during extreme intracellular acidosis, may reverse and couple the influx of H C 0 3 " to the efflux of CI" at both room temperature and at 37°C (also see Frelin et al, 1988). Other types of pHj regulating mechanisms have been characterized in other neuronal preparations but appear to play only a minor, i f any, role in pHj regulation in 14 hippocampal cells (Chesler, 1990). For example, Martinez-Zaguilan et al. (1994) reported that C17H+ co-transport activity was involved in pHj regulation in rat brain synaptosomes but no evidence could be found for its participation in pH, regulation in rat hippocampal neurons (Baxter and Church, 1996). Interestingly, in certain types of glial cells, the Na + /HC03" co-transporter appears to be the major pH; regulating mechanism (Deitmer and Schlue, 1987; Kettenmann and Schlue, 1988; Munsch and Deitmer, 1994; O'Connor et al, 1994). Again, N a + / H C 0 3 " co-transport appears not to contribute to the regulation of pHj in rat hippocampal neurons (Baxter and Church, 1996). Although a variety of pHj regulating mechanisms have been described in mammalian central neurons, the possibility that neurotransmitters can modulate the activity of these pHj regulatory systems has not been previously investigated. The evidence for this possibility originated, primarily, from studies involving non-neuronal preparations. Modulation of pH-t regulatory mechanisms by extracellular agents and second messengers In peripheral cell types, extracellular signals such as neurotransmitters, mitogens and hormones are able to alter the activities of pHj regulatory mechanisms. It appears that the cellular components mediating the coupling of extracellular agents to pHj regulatory mechanisms are tissue-specific and even, within the same tissue, receptor-specific. For example, serotonin is able to concurrently activate the N a + / H + exchanger 15 and the HCOy/Cl" exchanger in rat glomerular mesangial and vascular smooth muscle cells (Kahn et al, 1992; Saxena et al, 1993; Ganz and Boron, 1994) but, in rabbit ileal crypt and villus cells, serotonin again activates the N a + / H + exchanger but conversely, inhibits the HCOy/Cl" exchanger (Sundaram et al, 1991). In rat and guinea-pig cardiomyocytes, catecholamines such as norepinephrine and epinephrine induce an intracellular alkalinization via a i-adrenoceptors while R-adrenoceptors mediate a decrease in pHj (Iwakura et al, 1990; Gambassi et al, 1992; Guo et al, 1992; Wallert and Frohlich, 1992; Terzic et al, 1992; Lagadic-Gossman and Vaughan-Jones, 1993). However, activation of B-adrenergic receptors in canine enteric endocrine cells initiates an increase in pHj (Barber et al, 1992). The specific pHj regulating mechanisms involved in B-adrenergic receptor-mediated pHj modulation also exhibit tissue specificity. In most tissues, R-adrenoceptor agonists induce their effects on pHj via inhibition or acceleration of the activity of the Na+/H+ exchanger (Barber et al, 1992; Guo et al, 1992; Lee et al, 1994; Wu and Vaughan-Jones, 1994) but, in rat ventricular myocytes, P-adrenergic receptor agonists act primarily on the Na+-independent H C 0 3 7 C i " exchanger to produce a decrease in pHj (Desilets et al, 1994). The fact that a single extracellular agent, e.g. norepinephrine, can increase or decrease the activity of different pHj regulating mechanisms depending on the cell type leads to the conclusion that the cellular pathway(s) mediating these effects may also be cell- or tissue-specific. The interaction between receptor activation and pHj regulating mechanisms may also involve different intracellular second messengers, such 16 as cAMP (Guo et al, 1992; Wu and Tseng, 1993; Borensztein et al, 1993; Wu and Vaughan-Jones, 1994) or inositol 1,4,5 triphosphate (IP 3; Huang et al, 1987). For instance, in a wide variety of non-neuronal cell types, a rise in cytosolic [cAMP] can down-regulate or inhibit the activity of the N a + / H + exchanger (Guo et al, 1992; Wu and Tseng, 1993; Borensztein et al, 1993; Wu and Vaughan-Jones, 1994). The modulation by extracellular stimuli of the activity of pH, regulating mechanisms via a membrane-delimited pathway, that is a direct interaction between the receptor-activated heterotrimeric GTP-binding protein (G protein) subunit and pHj regulatory mechanism(s), has also been described (Barber et al, 1992; Dhanasekaran et al, 1994; Voyno-Yasenetskaya et al, 1994; Kitamura et al, 1995). Given that G-proteins and second messengers are involved in the alteration of the activity of pHj regulating mechanisms by external agents, the role of protein kinases and possible phosphorylation of pHj regulating mechanism(s) has also been investigated. Saxena et al. (1993) used glomerular mesangial cells to show that certain mitogens and serotonin increased the rate at which the N a + / H + exchanger restores pH; to steady state levels after an imposed intracellular acid load and found that this increased activity could be mediated by several isoforms of protein kinase C (PKC), one of which was the brain-specific isoform, y-PKC. Many authors have now determined that phosphorylation of the N a + / H + exchanger, initiated by growth factors and hormones, will influence the activity of the exchanger (e.g. Bianchini et al, 1991; Sardet et al, 1991; Winkel et al, 1993). In fact, Sardet et al. (1991) has postulated the existence of a "NHE-1 kinase(s)", activated by 17 growth factors, that would increase the activity of the N a + / H + exchanger by phosphorylation and Guizouarn et al. (1993) determined that the trout erythrocyte N a + / H + exchanger (RNHE) has two consensus sites on the cytoplasmic tail for phosphorylation by protein kinase A (PKA). And finally, Bertrand et al. (1994) identified the NHE-1 human isoform as a novel member of the calmodulin-binding proteins; its cytoplasmic domain strongly binds calmodulin in a calcium-dependent manner. This intracellular site on NHE-1 may participate in the activation of either the calcium-dependent/calmodulin-activated protein kinase (CaM KII) or the calcium/calmodulin-dependent protein phosphatase 2B, calcineurin. In this regard, Bianchini et al. (1991) utilized okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, to induce an increase in the activity of the N a + / H + exchanger in rat thymic lymphocytes. Therefore, the activity of the N a + / H + exchanger may depend on its level of phosphorylation. Different isoforms of the N a + / H + exchanger may even be present in the same cell type and be regulated by different signal transduction systems. For example, in renal and intestinal epithelia there seems to be a functional difference in the types of N a + / H + exchanger expressed in the apical and basolateral membranes of the same cell whereby the NHE-1 (basolateral) isoform of the N a + / H + exchanger does not possess a consensus sequence for cAMP-activated protein kinase (PKA) while the apically-located NHE-2 isoform can be activated by P K A (Borgese et al, 1994). A l l of the individual components mediating receptor-induced modulation of the activity of pHj regulating mechanisms have yet to be determined, but the diversity of 18 possible pathways is quite evident. For example, in renal brush border cells, Dj dopaminergic receptors are positively linked to adenylyl cyclase via activation of the G s isoform of G-proteins while, in pituitary lactotrophs, activation of D 2 receptor sub-types will , via the Gj subfamily of G-proteins, inhibit the activity of adenylyl cyclase which will result in decreased levels of intracellular cAMP. However, in these cell types, activation of D i - and D2-receptor sub-types mediate an inhibition of the N a + / H + exchanger that, while still G protein-mediated, is independent of changes in cytosolic [cAMP] (Ganz et al, 1990; Felder et al, 1993). Similar apparent discrepancies can clearly be seen in the study of Barber and Ganz (1992) in canine enteric endocrine cells. They concluded that norepinephrine, acting via P2 receptors, and somatostatin can accelerate and inhibit respectively, the activity of the N a + / H + exchanger, specifically the NHE-1 isoform. Interestingly, the cellular pathway mediating this effect did not involve the activation of G proteins G s and G , /G 0 and subsequent changes in cytosolic cAMP levels (see also Isom et al, 1987). Rather, norepinephrine and somatostatin were found to activate novel G proteins G^M G a i 3 which, in turn, modulated the activity of the N a + / H + exchanger in a membrane-delimited manner (Barber and Ganz, 1992; Voyno-Yasenetskaya et al, 1994). Finally, receptor-mediated changes in the activity of pH; regulating mechanisms may be consequent upon changes in [Ca 2 +]j evoked by calcium entry through ligand- or voltage-activated channels and/or released from intracellular stores (reviewed by Frelin et al, 1988). A n association between the cytoplasmic free C a 2 + concentration and the 19 activity of a variety of pHj regulating mechanisms has been observed in rat ventricular myocytes (Iwakura et al, 1990), vascular smooth muscle (Kikeri et al, 1990), pancreatic (3-cells (Juntti-Berggren et al, 1991), osteoblastic cells (Green and Kleeman, 1992), cardiac Purkinje fibers (Wu and Tseng, 1993) and a kidney cell line (Cano et al, 1994). And Sanchez-Armass et al. (1994) argued that calcium has an important role in the regulation of the N a + / H + exchanger in rat brain synaptosomes. This relationship, however, is not seen in all cell types (e.g. murine fibroblasts (Ives and Daniels, 1987) and rabbit ventricular myocytes (Ikenouchi et al, 1994)). To summarize, the intervening components involved in the modulation of the activity of pHj regulating mechanisms by surface receptor-mediated pathways appear to be tissue- and/or cell-specific. The causal effector of the intracellular pathways mediating surface receptor-mediated changes in the activity of a given pH-, regulating mechanism may be a G protein subunit, a protein kinase and/or a second messenger such as cAMP or Ca2+. Norepinephrine and the hippocampus As noted above, norepinephrine has been shown to affect the activity of pHj regulating mechanisms via intracellular second messengers in a variety of cell types. The present study investigated the possibility that norepinephrine may affect the activity of the acid extrusion mechanisms present in acutely isolated adult rat hippocampal CA1 neurons, a type of mammalian central neuron in which pHj regulating mechanisms have been well characterized. 20 Noradrenergic innervation of the adult rat hippocampal formation begins in the brainstem nucleus locus coeruleus (75 - 95% ipsilateral; Loy et al, 1980). Noradrenergic axons initially travel in the dorsal noradrenergic bundle, join the median forebrain bundle and enter the hippocampus by three pathways: the ventral amygdaloid bundle-ansa peduncularis, the ipsilateral fasciculus cinguli, and the fornix (Frotscher, 1990). The granule cell layer of the dentate gyrus receives the densest projection while the CA1 region of Amnion's Horn receives its modest innervation principally through the ipsilateral fasciculus cinguli (Loy et al, 1980). Although the CA1 region receives the lowest noradrenergic innervation of any hippocampal region, this area is associated with the highest receptor turnover rate and the modest innervation is offset by a higher rate of release of norepinephrine (Hortnagl et al, 1991). Loy et al (1980) used fluorescence histochemistry to substantiate that the noradrenergic axons form monosynaptic connections to pyramidal cells, particularly to the somata and dendrites of CA1 neurons. The effects of norepinephrine on neuronal function and the adrenergic receptor sub-types mediating the actions of norepinephrine in the hippocampal formation have been extensively documented {e.g. Cash et al, 1986; Nicoll et al, 1990). Autoradiographic localization studies have confirmed that each of the adrenergic receptor subtypes (ocl5 a 2 , p\ and p2) are located post-synaptically in the CA1 subfield of the adult rat hippocampus (Minneman et al, 1981; Booze et al, 1989; Duncan et al, 1991), although, in the CA1 region, there appears to exist a predominance of P, versus P 2-adrenergic receptor subtypes (Duncan et al, 1991). a2-adrenoceptors also seem to be localized on norepinephrine- and serotonin-releasing nerve terminals as autoreceptors as 21 well as on postsynaptic CA1 pyramidal neurons, whereas the a l a - and a ip-adrenergic receptors are located primarily postsynaptically (Segal et al, 1991; Zilles et al, 1991; Heal et al, 1993). Both subtypes of the P-adrenergic receptor are positively linked to adenylyl cyclase via the G s class of heterotrimeric G-proteins and lead to an increased production of intracellular cAMP. In contrast, a-adrenergic receptors, through activation of the G / G 0 subfamily of G-proteins, inhibit adenylyl cyclase and thus reduce the amount of cellular cAMP (Gilman, 1984; Limbird, 1988; Hille, 1992). Overview Many factors can influence pH ;, including pH 0 , neuronal activity and pathophysiological events. Interestingly, these induced changes in pHj can in turn modulate the events which initially caused them. Recently, the mechanisms that neurons rely upon to maintain strict control of their resting pH ; have started to be characterized in regions of the mammalian central nervous system. In peripheral cell types, external agents such as norepinephrine can also affect pHj by altering the activities of acid extrusion mechanisms. The possibility that similar processes may occur in mammalian central neurons has not, to date, been explored. In the present study, mechanisms regulating acid extrusion in isolated rat adult hippocampal CA1 neurons were first characterized and the actions of norepinephrine on steady state pH, and on the activity of acid extrusion mechanisms during recovery from an imposed intracellular acidification were then investigated. Finally, the intracellular signal transduction system(s) mediating the effects of norepinephrine on the N a + / H + exchanger were characterized. The dual-22 excitation ratio method was used to measure pHj employing the fluorescent hydrogen ion indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. 23 Materials and Methods Cultured post-natal rat hippocampal neurons Primary cultures of hippocampal neurons were prepared from 4 - 5 day old Wistar rat pups according to Zorumski et al. (1992). Transverse hippocampal slices (< 1 mm thick) were collected in ice-cold Leibovitz L-15 medium (pH 7.2 - 7.3 at 37°C; Gibco, Grand Island, N Y ) and then incubated for 30 minutes at 37°C in L-15 medium containing 0.2 mg/ml bovine serum albumin, 1.0 mg/ml papain (type IV; Sigma Chemical Co., St. Louis, MO) and 25 pg/ml DNAase (type II; Sigma Chemical Co.). Afterwards, the L-15 medium was discarded and replaced with a medium containing Eagle's minimal essential medium ( E M E M ; Gibco), 22 mM NaHC0 3 , 10 m M 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES), 17 mM glucose, 400 u M glutamine and 25 ug/ml DNAase dissolved in tissue culture grade water. The slices were mechanically dissociated immediately with fire-polished Pasteur pipettes of decreasing tip diameters, underlayed with fetal bovine serum (FBS) and then cold centrifuged at 150 x g at 4°C for 5 - 6 minutes. The supernatant was discarded and the pellet was re-suspended in E M E M . To determine the total viable cell concentration, a sample of the cell suspension was removed and mixed with trypan blue, a cell viability indicator. A hemocytometer (Neubauer) chamber was used to count the number of viable hippocampal neurons within the sample and a dilution factor was calculated so that the cell suspension could be diluted with E M E M , supplemented with 5% FBS and 5% horse serum (5/5 serum E M E M ) , to obtain a final density of 3.0 x 105 cells/ml. The neurons were then dispersed 24 onto 18 mm coverslips coated with poly-D-lysine and laminin and allowed to adhere for 1 - 2 hours in a 5% C 0 2 atmosphere at 37°C. The coverslips were transferred to six well plates and further incubated in 5/5 serum EMEM for 24 hours in a 5% C0 2 atmosphere at 37°C. The growth medium was then switched to a serum-free, N2-supplemented EMEM (containing 5 ug/ml insulin, 20 nM progesterone, 100 pM putrescine and 30 nM sodium selenite) and the cell cultures were treated with 10 uM cytosine-P-D-arabinofuranoside hydrochloride to suppress glial cell proliferation. The serum-free, N2-supplemented EMEM medium was replenished every 3-4 days. Each coverslip consisted primarily of hippocampal CA1 and CA3 neurons with a maximum of 15% of cells being glial and were used in experiments from 5-14 days after plating in N2-supplemented EMEM media. All neuronal cultures were provided by Ms. Stella Amatjda, Department of Physiology, University of British Columbia. Acutely dissociated adult rat hippocampal CA1 neurons Acutely dissociated adult rat hippocampal CA1 neurons were utilized for the majority of the studies. Adult rat hippocampal CA1 neurons were isolated using a modified version of the procedure described by Kohr and Mody (1991). Male Wistar rats approximately 45 days old (200 - 240 g) were obtained from the Animal Care Center (University of British Columbia) and housed under conditions of controlled temperature (20°C - 22°C) and lighting (lights on 0600-1800 h). Food (Lab Diet, PMI Feeds Inc., St. Louis, MO) and water were available ad libitum. The animals were anesthetized with 3% 25 halothane in air, rapidly decapitated, and their brains were rapidly removed and placed in ice-cold (4°C - 8°C) HC0 37C0 2-buffered saline (Table 2, solution 6) previously equilibrated with 5% C0 2 /95% 0 2 . One of the hippocampi was separated from the surrounding tissue as outlined by Tyler (1980) and transverse hippocampal slices, 450 um thick, were obtained with a Mcllwain tissue chopper and collected in ice-cold (4°C -8°C) HC037C0 2-buffered medium. The slices were allowed to recover for at least one hour in an incubation chamber at 32°C in HC0 37C0 2-buffered saline. Three hippocampal slices were then enzymatically digested for 30 minutes at 32°C in 2.0 ml of HC0 37C0 2-buffered saline containing 1.5 mg/ml of pronase (protease type XIV bacterial from Streptomyces griseus; Sigma Chemical Co.). The CA1 regions of each slice were then microdissected with a dissecting chisel and triturated with fire-polished Pasteur pipettes of diminishing tip diameters (0.7, 0.5, 0.3 and 0.2 mm) in 0.5 ml of HEPES-buffered saline (Table 1, solution 1), pH 7.35 at room temperature. The triturated mixture was then deposited onto an 18 mm poly-D-lysine- (20-50 ug/ml; Sigma Chemical Co.) coated glass coverslip. The neurons were allowed to adhere to substrate for 15 minutes at room temperature exposed to a 100% 0 2 atmosphere. Each trituration produced ~10 - 20 viable hippocampal CA1 neurons free from cellular debris, although the pHiS of only 1 -3 neurons were measured simultaneously in any given experiment. Freshly isolated hippocampal CA1 neurons were chosen for study based on morphological criteria established by Schwiening and Boron (1994), i.e. smooth, non-granular appearance; a single major process (presumably an apical dendrite) projecting from one pole of the 26 soma which was at least twice the length of the diameter of the cell body; and the presence of two or more smaller processes at the opposite pole. Cell loading and BCECF The microspectrofluorimetric technique was used to measure pHj employing the intracellular fluorescent hydrogen ion indicator, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Punk et al., 1982). The membrane permeant acetoxymethyl ester of BCECF (BCECF-AM; Molecular Probes, Inc., Eugene, OR) was prepared initially as a 1.0 m M stock solution in anhydrous dimethylsulphoxide (DMSO) and stored at -60°C. B C E C F - A M , being hydrophobic and uncharged, can pass easily through the plasma membrane and, upon entry into the neurons, is hydrolyzed by intracellular esterases to produce the hydrophilic, polyanionic B C E C F free acid which is trapped within the neuron. On the day of an experiment employing cultured post-natal hippocampal CA1 neurons, a loading medium was prepared consisting of HEPES-buffered saline, pH 7.35 at room temperature (Table 1, solution 1), with the isoosmotic addition of 3.0 m M N a H C 0 3 replacing NaCl. When needed, 5.0 pl of the 1.0 m M B C E C F - A M stock solution was dissolved into 2.5 ml of the loading medium to achieve a final concentration of 2 u M B C E C F - A M . A coverslip with post-natal neurons attached was placed face-up in the 2 u M BCECF-AM-containing loading medium for 30 minutes at room temperature. The coverslip was then transferred onto an insert of the perfusion/recording chamber, supported by a 22 mm glass coverslip and then sealed with an O-ring rimmed 27 with high vacuum silicone grease (Dow Corning, Midland, MI) so as to form the floor of the chamber. For each experiment involving acutely dissociated hippocampal CA1 neurons, 1.0 pl of 1.0 mM B C E C F - A M stock solution was pipetted gently onto the plated neurons (which were contained in 0.5 ml of HEPES-buffered saline; see above) and the neurons were allowed to load with 2 u M BCECF for 15 minutes at room temperature in a 100% 0 2 atmosphere. For experiments measuring changes in intracellular free Ca 2 + concentration ([Ca2+]j), the fluorescent indicator fura-2-AM (5 u M ; Grynkiewicz et al., 1985; Molecular Probes, Inc.) was loaded into the neurons in the same manner as BCECF, except the loading occurred at 37°C for 60 minutes. For both cultured post-natal hippocampal neurons and acutely dissociated adult hippocampal CA1 neurons, the prepared chamber (containing neurons loaded with BCECF or fura-2) was placed onto the stage of the microscope. The neurons were then superfused continuously with HEPES-buffered saline (Table 1, solution 1), pH 7.35 at 37°C, at a rate of 2.4 ml/min for 15 minutes prior to the start of each experiment as the temperature of the perfusate within the chamber was slowly increased to 37°C. The temperature-controlled perfusion chamber allowed for relatively uninterrupted and rapid switching of perfusion solutions, e.g. HEPES-buffered to HC0 37C0 2-buffered medium. When perfusing with HC0 37C0 2-buffered media, the atmosphere in the perfusion chamber contained 5% C 0 2 in balance air. 28 Experimental setup and the ratiometric method The dual-excitation ratio method for estimating pHj with B C E C F is based upon the relationship between pH and the ratio of emitted fluorescent intensities at alternating wavelengths of excitation at 488 nm and 452 nm. Fluorescence emission during excitation at 488 nm is pH sensitive whereas emission during excitation at 452 nm is relatively pH insensitive. For fura-2, also a dual-excitation fluorescent dye, the excitation wavelengths employed were 334 nm and 380 nm, both of which are sensitive to changes in [Ca2 +] ;. Because the emitted intensities at both excitation wavelengths are from the same cell volume, a ratio of intensities emitted at two different excitation wavelengths will , in principle, not be susceptible to artifacts caused by variations in optical path length, local probe concentrations, illumination intensity and photobleaching (Bright et al, 1987; Brighter al, 1989). Throughout an experiment, the viability of the neurons was assessed by monitoring the stability of the fluorescence emission during excitation at 452 nm (/ 4 5 2; pHpinsensitive). Bevensee et al. (1995) monitored the rates of B C E C F loss and morphological deterioration of acutely isolated post-natal hippocampal CA1 neurons and determined that the rate of change of I452 could be used as a sensitive indicator of cell membrane integrity. In addition, studies from the same laboratory indicate that acutely dissociated rat hippocampal neurons which retain BCECF also exclude propidium iodide, a widely-employed indicator of cell membrane integrity (Bevensee et al, 1995). The experimental apparatus consisted of a Zeiss Axiovert 10 microscope (Carl Zeiss Canada Ltd., Don Mills, ON) in conjunction with an Attofluor Digital Fluorescence 29 Ratio Imaging System (Atto Instruments Inc., Rockville, MD). U V light, emitted from a 100 W mercury arc lamp, was alternately passed through one of two 10 nm interference filters, centered at 488 nm and 452 nm, and was then reflected by a long band-pass dichroic mirror (FT > 495 nm) into the objective (Zeiss Achroplan, n.a. 0.60, 40x) for excitation of the pH indicator loaded in the neurons. Fluorescence emitted by the neurons (at 520 nm) was passed back through the objective, the dichroic beam splitter and a 510 nm barrier filter to be measured by an intensified charge-coupled device camera. The camera gain was set by maximizing the image intensity while minimizing the possibility of camera saturation, and was held constant throughout an experiment. The images were digitized to an 8 bit resolution with a 512 x 480 pixel frame size. Data were obtained from multiple neuronal cell bodies simultaneously, with each neuron delineated as a region of interest (ROI). In order to prevent photobleaching of the dye and UV-induced damage to the neurons, neutral density filters were placed in the U V light path to reduce the intensity of light transmitted at each wavelength. In addition, the light path from the mercury U V source was interrupted by a computer actuated high speed shutter restricting the exposure of the neurons to the U V light to periods only when emitted fluorescence intensities at each excitation wavelength were being measured (usually once every 2 to 15 seconds). Finally, a variable intensity lamp control (Attoarc, Carl Zeiss Canada Ltd.) was employed to reduce the intensity of 100 W mercury arc lamp by 50%. 30 Solutions and chemicals A l l experiments were performed at 37°C. The HEPES-buffered and H C 0 3 7 C 0 2 -buffered media employed in experiments utilizing acutely dissociated neurons are listed in Tables 1 and 2, respectively. For experiments involving cultured post-natal hippocampal neurons, the solutions were identical to those listed in Tables 1 and 2 except for the concentration of glucose which was reduced to 10 mM from 17.5 mM. The N a + free, HEPES-buffered medium in which L i + was substituted for N a + was prepared by replacement of NaCl and NaH 2 P0 4 (Table 1, solution 1) with 138 m M L i C l . A l l solutions were prepared at room temperature. Accordingly, HEPES-buffered solutions were titrated to pH 7.48 - 7.50 (at room temperature) in order for the media to be pH 7.35 - 7.36 at 37°C. The pH to which the HEPES-buffered media were titrated, at room temperature, was determined by the equation p H 3 7 = 0.18 + 0.96 x p H R T , (Equation 1) where p H 3 7 denotes the pH of the HEPES-buffered saline at 37°C and p H R T represents the titrated pH of the HEPES-buffered media at room temperature (Baxter, 1995). To vary the pH of HC03"/C02-buffered solutions, the concentration of N a H C 0 3 was adjusted (at a constant -PC02) according to the equation p H 3 7 = 6.03 + 1.03 x log[HC0 3], (Equation 2) where p H 3 7 represents the pH of a HC037CC»2-buffered solution (~ pH 7.35 - 7.36) at 37°C following equilibration with 5% C 0 2 (Baxter, 1995). A l l HEPES-buffered 31 solutions were titrated with 10 M NaOH except for the high-K + calibration medium (Table 1, solution 5) and the Na+-free salines substituted with N M D G + (Table 1, solution 2) or L i + , which were titrated with 10 M KOH, 1.0 M HC1 and 2.0 M LiOH, respectively. A Corning 240 pH meter, calibrated daily, was utilized to measure the pHs of all solutions. A l l stock solutions, except for DIDS, were prepared in advance and stored at -60°C. Nigericin, an antibiotic from Streptomyces hygroscopicus (Sigma Chemical Co.), was stored as a 10 m M stock solution in ethanol. On the day of an experiment 100 pl of the nigericin stock was thawed and diluted to 10 uM in high-K + HEPES-buffered saline, pH 7.00 at 37°C. Norepinephrine and all other adrenergic receptor agonists and antagonists were dissolved in ultrapure distilled water (Milli-Q UF Plus, Millipore Ltd., Mississauga, ON) and stored as 50 m M stock solutions with 5.0 m M sodium ethylenediaminetetraacetate (NaEDTA). On the day of the experiment, stock solutions were dissolved in physiological media to the desired test concentration with 0.3 m M ascorbic acid. NaEDTA and ascorbic acid were employed to delay the oxidative degradation of the adrenergic receptor agonists and antagonists, especially important at 37°C (Hughes and Smith, 1978). EIPA was initially prepared as a 50 mM stock solution in DMSO and then further diluted to 50 uM in physiological media. Forskolin, 2',5'-dideoxyadenosine, l',9'-dideoxyforskolin and 3-isobutyl-l-methylxanthine (IBMX) were dissolved in DMSO to a stock concentration of 50 mM, while N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was stored at -60°C as a 10 m M stock solution in DMSO. Sp-cAMPS and Rp-cAMPS were dissolved in ultrapure 32 water and stored at 25 u M and 50 uM, respectively. Stock solutions for cholera toxin were prepared either at 50 uM, for experiments involving acute exposure of the toxin, or at 500 Lig/ml, for experiments that entailed long term incubation with the G s a activator. DIDS was prepared on the day of the experiment as a 100 m M stock in DMSO and further diluted to the appropriate test concentration using physiological saline. Appropriate control experiments were performed involving DMSO, NaEDTA and ascorbic acid and the results indicated that these agents evoked no significant changes in either steady state pHj or on the rate of recovery of pHj from an imposed intracellular acidification (data not shown). The final concentration of DMSO in the test solutions never exceeded 0.1%. Pharmacological agents were purchased from Research Biochemicals International (Natick, M A ) except for: cholera toxin, terbutaline and DIDS (Sigma Chemical Co.); 2',5'-dideoxyadenosine and H-89 (Biomol Research Laboratories Inc., Plymouth Meeting, PA); and Rp-cAMPS and Sp-cAMPS (Biolog Life Science Institute, La Jolla, CA). Experimental paradigms The effects of pharmacological treatments and of extracellular ion manipulations (e.g. L i + substituting for external Na +) on steady state pHj were examined. At resting (physiological) pHj, pHi regulating mechanisms present may or may not be active. For example, in cultured fetal rat hippocampal neurons, it has been shown that the N a + -independent HC0 3 7Cr exchanger is operational as an acid extrusion mechanism only at 3 3 very low levels of pHj (pHj < 6.4) and not at "normal" resting pHj values (Baxter and Church, 1996). Therefore, the effects of pharmacological and other treatments were also assessed on the rate of recovery of pHj after an imposed intracellular acid load. Intracellular acidification was induced by the N H 4 + prepulse technique (Boron and De Weer, 1976). This involves exposing the neurons to a medium containing the weak base N H 4 + (Table 1, solution 4 and Table 2, solution 9), a maneuver which causes resting pHj to alkalinize. Upon washout of the NH 4 +-containing physiological saline, pH, undershoots its initial steady state value. From this acidified level of pHj, the neuron utilizes acid extrusion mechanisms to recover to the normal resting level of pHj (Roos and Boron, 1981; Boron, 1989). In each acid load recovery experiment, two acid load maneuvers were performed. The initial acid load was used to calculate the internal control rates of pHj recovery for a given neuron after an imposed acid load while the second acid load and subsequent recovery of pH ; was performed under the influence of a pharmacological or other treatment. The control rates of pHi recovery were compared to the rates of pHj recovery in the presence of drug at the same absolute values of pHi. The rationale for such a comparison is based on the observation that the acid extrusion rate is greatest at the point of maximum acidification after an N H 4 + prepulse and gradually falls as pH, returns to steady state levels (see Boyarsky et al., 1988). Since the activity of acid extrusion mechanisms is determined, at least in part, by the absolute level of pH i ; by comparing rates of pHj recoveries at the same absolute values of pHj, the influence of pHj on the 34 activities of acid extrusion mechanisms (and, thus, rates of recovery of pH,) will be accounted for. Calculation ofpHtand analysis of data At the beginning of each experiment, background readings of autofluorescence at each excitation wavelength were measured in an area devoid of cellular elements. Using Visual Basic macros running in Microsoft Excel 5.0 (Microsoft Corp.), the respective background fluorescence intensities were subtracted from the raw intensities at each wavelength from each ROI throughout the experiment to yield background-corrected ratios (BIm/BI452). At the end of each experiment, a one-point calibration was performed where the neurons were exposed to an high-K+ HEPES-buffered calibration solution, pH 7.00 at 37°C (Table 1, solution 5) that contained 10 p M nigericin, a K + / H + ionophore (Thomas et al., 1979). Nigericin, a charged electron carrier, balances the ionic concentrations of cytoplasmic and extracellular K + and, in doing so, equilibrates p H 0 to pHj (Chaillet and Boron, 1985). Since external pH was titrated to pH 7.00, pH ; was eventually clamped at pH 7.00 after 5-10 minutes of exposure to the calibrating solution. For each experiment, the average background-corrected ratio of emitted intensities during the calibration period represented the normalizing ratio corresponding to pH s 7.00. The experimentally-derived background-corrected ratios (BI4$%/BI452) were then divided by the normalizing ratio corresponding to pH 7.00 and converted to corresponding values of pH; using Equation 3, which is based on the Henderson-Hasselbalch expression for the dissociation of a weak acid pHj = log[(Rn - R n ( m i n ) ) / (Rn(max) - R n ) l + pKa, (Equation 3) 35 where R n denotes the background-corrected normalized ratio being converted to pH;, R.n(max) and R n ( m i n ) represent the maximum and minimum obtainable values for the normalized ratio, and pKa is the -log of the dissociation constant of BCECF (for the derivation of Eq. 3 see Baxter, 1995). R n ( m a x ) , Rn(min) and pKa were derived from in situ calibration experiments (Fig. 1). In such experiments, the neurons were exposed to a series of high-K + HEPES-buffered solutions, each containing 10 uM nigericin, titrated to pHs ranging from ~ pH 5.5 to pH 8.5 in 0.5 pH unit increments using 10 M K O H or 1 M HC1 (Fig. 1A). The average background-corrected ratios (BIm/BI452) for all neurons at each pH were normalized with the average ratio of emission intensities at pH 7.00 and a pH titration curve was produced (Fig. IB). The upper and lower asymptotes of this curve represent the R n ( m a x ) and R n ( m i n ) , respectively, while the cytosolic pH on the x-axis corresponding to the point of inflection of the curve represents the measured pKa of B C E C F . The advantage of this normalization procedure is that it allows for a one-point calibration for each neuron or coverslip (i.e. population of neurons) examined. The calibration parameters were not affected by the temperature of the perfusate or by the age of hippocampal neurons and were reassessed whenever the mercury arc burner was replaced or the optical setup of the microscope was altered. For the 7 calibration experiments used in analyzing all experiments, the values for R n ( m a x ) , R n(min) a n d pKa (mean ± s.E.M.) were 2.11 ± 0.02, 0.46 ± 0.03 and 7.31 ± 0.02, respectively. Changes in resting pHj or in the rates of pHj recovery from an imposed acid load evoked by pharmacological treatments or external ion manipulations were analyzed with 36 paired two-tailed Student's t tests. Errors are expressed as S.E.M. For each adult hippocampal neuron, the average resting pH-, was determined before and after the neuron was exposed to the particular pharmacological treatment or external ion manipulation. The initial value of resting pHj and the resultant alkalinized or acidified level of steady state pHj were compared. Any net difference in steady state pH s was deemed to be significant i f the resultant P value was < 0.05. Using the other experimental paradigm, recovery from an imposed intracellular acid load, the recovery portion of the experiment was fitted, utilizing the least squares method (SigmaPlot v. 1.02, Jandel Scientific, San Rafael, CA), to a single exponential function of time having the format ( Ct) pHj = a + b(l-ev" ;), Equation 4 where a, b, and c are the exponential parameters. The differentiated form of Equation 4 yields the rate of change in pHj dpHj _ c t ^ = bee , Equation 5 from which the instantaneous rates of recovery (dpH/dr) were calculated at 0.05 unit intervals of pHj from the point of maximum acidification following an N H 4 + prepulse until recovery was ~ 75% complete (see Baxter and Church, 1996). For each neuron, a paired experiment, which consisted of two consecutive intracellular acid loads and subsequent pH; recoveries, was performed. The initial 37 intracellular acid load was used to determine the internal control rates of pHj recovery while the rates of pHi recovery following the second acid load were under the influence of a pharmacological treatment. The control rates of pHi recovery were compared to the rates of pH; recovery under the influence of a pharmacological treatment at all corresponding absolute values of pH s (at 0.05 pH unit increments from the point of maximum acidification until recovery was ~ 75% complete). At each corresponding absolute value of pH i ; the percentage difference between the control rate of pH; recovery and the rate of pH ; recovery in the presence of a drug was determined. An average of the resultant percentage differences was calculated and utilized for description of the data (see Results). To determine the intrinsic percentage differences in rates of pH, recovery between two consecutive acid loads, paired experiments were performed under control conditions, i.e. the second acid load and subsequent pH; recovery was also performed under control conditions. Based on data from 11 neurons, the maximum difference observed in the rates of pH; recovery from the second acid load compared to the first acid load, at any given absolute value of pHj, was a 20 ± 20 % increase (20 ± 66%, mean ± S.D.; Fig. 2). Therefore, rates of pHj recovery under the influence of a pharmacological treatment were considered to be different from the control rates of pHi recovery only if they displayed an average increase of 86% or more, as compared to the control rates of pH ; recovery. Only neurons which exceeded this arbitrarily-imposed criterion were considered to have responded to the pharmacological treatment and, as such, only data from these neurons underwent further analysis. 38 In addition, for each neuron, a formal statistical comparison was made between the instantaneous rates of pH-, recovery (evaluated at 0.05 units of pH, from the point of maximum acidification until recovery was ~ 75% complete) under control conditions and those under the influence of a pharmacological treatment. The instantaneous control rates of pHj recovery and the rates of pH, recovery in the presence of a pharmacological treatment were grouped separately and a paired Mest was employed to assess statistical difference. If the resultant P value was < 0.05, then the rates of pHj recovery from an imposed intracellular acid load were considered to have been altered significantly by the pharmacological treatment. In summary, if the criteria for the average percentage difference between "control" and "treatment" acid load recoveries were satisfied and i f the statistical comparison between the grouped instantaneous rates of pHj recovery under control conditions and those in the presence of drug returned a P value < 0.05, then the increased rates of pHj recovery are most likely due to a statistically relevant enhancement of the acid extrusion rate by the test compound. From experiments involving acutely dissociated adult hippocampal neurons, the resultant traces (pH; vs. time) depict data collected from a single adult rat hippocampal CA1 neuron and n values refer to the number of individual neurons from which data were obtained. In contrast, pHj vs. time graphs generated from experiments involving cultured hippocampal neurons represent the mean of data obtained simultaneously from all viable neurons on each coverslip. In experiments employing cultured neurons, the n value refers to the number of cell populations (i.e. number of coverslips) analyzed. The data 39 generated from experiments employing fura-2 are presented as background-corrected ratios of emission intensities (BI33JBI3S0), i.e. the ratios were not calibrated. Periodically, the 100 W mercury arc burner produced brief fluctuations in the intensity of the U V light output which resulted in variations in the emission intensities and ultimately, the values of pHj. In order to smooth the graphical representation of the pHj vs. time records, a moving average (period = 3) was applied to all traces shown (see Boyarsky et al, 1988; Baxter and Church, 1996). 40 Table 1: Composition of HEPES-buffered experimental solutions (all concentrations in mM): Solution 1 2 3 4 5 Standard N a + free Ca 2 + free NH 4 C1 High K + NaCl 136.5 - 136.5 116.5 -KC1 3.0 3.0 3.0 3.0 -C a C l 2 2.0 2.0 - 2.0 2.0 N a H 2 P 0 4 1.5 - 1.5 1.5 1.5 M g S 0 4 1.5 1.5 3.5 1.5 1.5 Na Glu - - - - 10.0 K G l u - - - - 130.5 D-glucose 17.5 17.5 17.5 17.5 17.5 NMDG+ - 136.5 - - -NH 4 C1 - - - 20.0 -HEPES 10.0 10.0 10.0 10.0 10.0 Titrated 1 0 M 10M 10 M 1 0 M 10 M with: NaOH HC1 NaOH NaOH K O H Ca2+-free medium also contained 200 uM ethylene glycol-bis(R-aminoethyl ether) N, N, JV, JV-tetraacetic acid (EGTA). Abbreviations: Na Glu, sodium gluconate; K Glu, potassium gluconate; NMDG+, A^-methyl-D-glucamine. 41 Table 2: Composition of HC037C02-buffered experimental solutions (all concentrations in mM): Solution 6 Standard 7 N a + free 8 CI" free 9 NH4CI NaCl 126.5 - - 106.5 N a H C 0 3 20.0 - 20.0 20.0 KC1 3.0 3.0 - 3.0 C a C l 2 2.0 2.0 - 2.0 N a H 2 P 0 4 1.5 - 1.5 1.5 M g S 0 4 1.5 1.5 1.5 1.5 D-glucose 17.5 17.5 17.5 17.5 N H 4 C I - - - 20.0 Na Glu - - 126.5 -K G l u - - 3.0 -V^Ca Glu - - 4.0 -Choline H C 0 3 - 20.0 - -Choline CI - 126.5 - -A l l HC0 3 --containing solutions were equilibrated with 5% C 0 2 in balance air. Abbreviations: Na Glu, sodium gluconate; K Glu, potassium gluconate; V^Ca Glu, hemi-calcium gluconate. 42 Figure 1. Sample calibration plot for BCECF A. Cultured post-natal neurons were exposed to HEPES-buffered medium containing 10 u M nigericin at pH 0 (and therefore pHj) 5.55, 6.03, 6.50, 7.00, 7.49, 7.99 and 8.48. The duration of each exposure is indicated by the bars above the trace, which is a mean of data obtained from 13 neurons recorded on a single coverslip. The resultant background subtracted ratios (I^JI^i) were normalized to 1.00 at pH; 7.00. B. Plot of pH ; against the resulting normalized ratios (RJ. Standard error bars are indicated (n = 3 coverslips). The curve is a result of a non-linear least squares regression fit to Equation 3. For this particular calibration, the values of R,,^^, R„( m in) and p/Ca were 2.05, 0.48 and 7.24, respectively. 43 44 Figure 2. Control acid load recoveries during perfusion with HEPES-buffered medium A . In order, in paired experiments, to determine whether a pharmacological treatment has an effect on the rates of pHj recovery from an imposed intracellular acidification as compared to the internal control rates of pH; recovery, the intrinsic differences between the rates of pH; recoveries of two consecutive acid loads must be accounted for. In the example shown, an initial acid load was performed and, after pH; had recovered fully, a second acid load was conducted. The percentage differences between the rates of pHj recovery of the initial acid load and the rates of pHj recovery of the second maneuver at each corresponding level of absolute phi. were calculated. An average of the percentage differences was then determined. In this case, the rates of pHj recovery from the second acid load were on average 60% slower than the rates of pH; recovery from the initial acid load. Shown also is a one point calibration with 10 uM nigericin at pH 7.00. B. The graph represents a comparison between all of the rates of pH, recovery from an initial (A) and from a second (•) acid load, collected from 11 neurons at the same absolute values of pHi. Both sets of data were independently fitted with a linear least-squares regression (r2= 0.85 (A) and 0.87 (•)). The maximum difference in the rates of pH; recovery from the second acid load as compared to the rates of pH, recovery from the initial acid load was a 20 ± 20% (20 ± 66%, mean ± S.D.) increase at pPf 7.1. 45 46 Results The present study first characterized the acid extrusion mechanisms present in acutely dissociated adult rat hippocampal CA1 neurons, and then proceeded to determine the effects of norepinephrine on steady state pH; and on the rate of pH; recovery after an imposed intracellular acidification. The intracellular pathway mediating the ability of norepinephrine to alter the activity of one of the acid extrusion mechanisms found to be present, the Na7H + exchanger, was then characterized. Except where noted, all experiments were performed with acutely dissociated adult rat hippocampal CA1 neurons. A) Characterization of acid extrusion mechanisms In H C 0 3 7 C 0 2 -buffered saline (pH 7.36 at 37°C), the average resting pH, for 55 neurons was 7.20 ± 0.03 and in HC0 3-free, HEPES buffered medium (pH 7.35 at 37°C), 439 neurons had an average resting pHi of 7.29 + 0.01. In both HC03~-free, HEPES-buffered medium and HCOy/CO^ -buffered saline, the values of resting pH, had a broad range (pH; 6.6 - 7.9 and pH; 6.6 - 7.5, respectively), as illustrated in Figure 3. The distribution of steady state pH, values for the neurons during perfusion with H C G y / C O ^ -buffered saline was best fitted by a single Gaussian distribution (Fig. 3B). However, the distribution of the resting pHj values of all neurons perfused with HC03"-free, HEPES-buffered saline was best fit with the sum of two Gaussian distributions with means of pHj 6.91 ± 0.01 and pH ; 7.43 ± 0.01 (Fig. 3A). 47 i) Acid extrusion during perfusion with HCOj-free, HEPES buffered medium In a nominally HC03"-free, HEPES buffered medium, any contribution of HCCy-dependent mechanisms to acid extrusion and the maintenance of steady state pH ; will be excluded. Initial studies employing a potent inhibitor of Na7H + exchange in other cell types, 5-(/V-ethyl, iV-isopropyl)-amiloride (EIPA), indicated that the amiloride analogue had no effect on steady state pHi (n = 3; Fig. 4) or on the rate of pH; recovery from an induced intracellular acid load (n = 4; Fig. 4). However, exposing the neurons to a Na + -free medium (substituted by NMDG + ) evoked an average decrease in steady state pH ; of 0.30 ± 0.03 pH units (n = 15; Fig. 5A) and blocked recovery of pH; from an imposed intracellular acid load (n = 5; Fig. 6A). In contrast to N M D G + , L i + can act as a substrate for the N a + / H + exchanger (Aronson, 1985). Accordingly, in the next series of experiments, external Na + was replaced by L i + and when applied, resting pHj decreased transiently but recovered in the continued absence of extracellular Na + (n = 4; Fig. 5B). Although L i + has a higher affinity than Na + for the Na7H + exchanger, its rate of translocation across the membrane is much slower which may explain the initial transient decrease in pH ; (Aronson, 1985; also see Baxter and Church, 1996). Similarly, when Na+-free, Li+-substituted medium was applied at the point of maximum acidification following an N H 4 + prepulse, pH, recovery could still occur (n = 6; Fig. 6B). The results indicate that under nominally HC03"-free, HEPES-buffered conditions, both resting pH, and the recovery of pH, following an imposed acid load are governed by a HC03"-independent, Na+-dependent mechanism that can also transport 48 external L i + in exchange for intracellular protons. This mechanism is likely to be a Na7H + exchanger which, unusually, is resistant to a potent analogue of amiloride, EIPA. A n amiloride-insensitive variant of the Na7H + exchanger has previously been described in adult, neonatal and fetal rat hippocampal neurons by a number of laboratories (Raley-Susman et al, 1991; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996). ii) Mechanisms of acid extrusion in the presence of HCOj To determine whether HCOy-dependent mechanisms could also participate in the maintenance of steady state pH„ initial experiments examined the changes in steady state pHj during the transition from a HCOy-free, HEPES buffered saline to a HCOy-containing medium, at a constant pH 0 . Upon exposure to HC0 37C0 2-buffered medium, an increase in steady state pH; typically occurred, the magnitude of the increase being dependent upon the initial resting level of pH; in HEPES buffered saline. As seen in Figure 7A, the addition of HCOy to the perfusion medium caused the resting pHj of an hippocampal CA1 neuron (initial pH; of 6.86) to increase by ~ 0.34 pH units. In contrast, Figure 7B depicts a neuron with a higher level of steady state pH : during perfusion with HC0 37C0 2-free, HEPES-buffered medium (initial pH, of ~ 7.23) which experienced only a very minor increase in steady state pHj upon the transition to a HC03"-containing perfusate. The effects of the transition from HC03"-free to HC03"-containing medium on steady state pHj in 20 neurons are shown in Fig. 7C. Further experiments were performed on neurons with initial resting pH; < 7.3 and determined that the anion exchange inhibitor, 49 DIDS, blocked the increase in resting pH; observed upon the addition of HC0 3 " to the perfusate in 6 out of 6 neurons tested (Fig. 8A). These results suggest that a H C 0 3 -dependent, DIDS-sensitive mechanism contributes to the maintenance of steady state pH ; for neurons with a resting pH, < 7.3. Upon removing external CI", resting pH; increased by an average of 0.15 ± 0.02 pH units (n = 8; Fig. 8B). However, as shown in Figure 8B, i f a neuron was exposed to a Cl"-free medium containing 300 u M DIDS, the previously observed alkalinization of steady state pH; was markedly attenuated (n = 5). The 0 [Cl"]0-evoked intracellular alkalinization observed in the presence of HC0 3 " was only observed in neurons with a resting pH ; < 7.3 (data not shown). Taken together, these results suggest that the H C 0 3 -dependent, DIDS-sensitive mechanism which contributes to the maintenance of resting pHj in acutely dissociated hippocampal CA1 neurons may be a HC037C1" exchanger. In the next series of experiments, neurons were exposed to a Na+-free, N M D G + -substituted medium in the presence of HC0 3". This maneuver should block the Na7H + exchanger and also the HC0 3 /C1" exchanger described above, i f it is a Na+-dependent isoform of the anion exchanger. As illustrated in Fig. 9A, exposure to Na+-free, N M D G + -substituted medium evoked an average decrease in resting pH; of 0.35 ± 0.10 pH units (n = 3). This decrease in resting pHj showed no signs of recovery in the continued absence of external Na + . Similarly, as shown in Fig. 9B, exposure of a neuron to Na+-free, NMDG+-substituted medium, at the point of peak intracellular acidification following an NH4+-prepulse, completely abolished pHj recovery. These results suggest that the DIDS-sensitive HCOy/Cl" exchanger present in rat hippocampal CA1 neurons is Na+-dependent, 50 as previously reported by Schwiening and Boron (1994), Baxter and Church (1996) and Bevensee et al. (1996). To summarize, under HC0 37C0 2-free, HEPES-buffered conditions, rat hippocampal CA1 neurons utilize an amiloride-insensitive variant of the Na7H + exchanger to extrude protons following an imposed intracellular acid load and to maintain pH; at normal resting levels. Under HCOy-containing conditions, this acid extrusion mechanism is supplemented by the activity of a DIDS-sensitive, Na+-dependent HC0 3 " ICY exchanger, the activity of which is dependent upon the absolute level of pH;. These results are entirely consistent with the literature concerning acid extrusion mechanisms in rat hippocampal neurons (Raley-Susman et al, 1991; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996). B) Effects of norepinephrine on pH, In HCO37CO2 -buffered saline, application of 10 u M norepinephrine increased steady state pHj, after a short delay ( - 3 - 5 minutes), by an average of 0.20 ± 0.02 pH units in 6 out of 7 neurons tested (Fig. 10A). In the remaining neuron, norepinephrine had no effect on steady state pHj. Norepinephrine also increased the rate of pHj recovery from an induced intracellular acidification in 5 out of 5 neurons examined by an average of 193% (Fig. 10B). As outlined in the Materials and Methods, the average percentage increase in the rate of pH s recovery represents the mean of the percentage increases in the rates of pH; recovery under the influence of a pharmacological treatment when compared to the control rates of recovery, measured at all corresponding absolute values of pHj. 51 The same experiments were repeated under nominally H C 0 3 -free, HEPES-buffered conditions and norepinephrine evoked an average rise of resting pH; of 0.27 ± 0.01 pH units in 13 out of 15 neurons tested (Fig. 11 A). In the remaining two neurons, norepinephrine had no effect on resting pHj. The effect of norepinephrine on steady state pH, persisted even after the norepinephrine application was terminated and for as long as stable recordings could be maintained (~ 30 minutes; n = 6; Fig. 12). Norepinephrine (10 uM) also increased the rate of pH ; recovery from an imposed intracellular acidification under HC0 37C0 2-free, HEPES-buffered conditions by an average of 161% (n = 15/17; Fig. 1 IB). In the remaining two neurons, the net effects of 10 u M norepinephrine on the rates of pH; recovery were a 31% decrease and an 11% increase, respectively. In 15 paired experiments of the type shown in Fig. 1 IB, the rates of pHj recovery from acid loads imposed under the influence of 10 u M norepinephrine were compared to the corresponding control rates of p H recovery at the same absolute values of pHj in HEPES-buffered saline (see Materials and Methods). The resulting plots of the rate of pH ; recovery vs. absolute level of pHj in both the absence and presence of 10 u M norepinephrine are shown in Fig. 13. Norepinephrine significantly increased the rates of pHi recovery from imposed intracellular acid loads when compared to rates of pH ; recovery from control acid loads (P < 0.05 at all absolute values of pH ;). To date, there is no known inhibitor for the amiloride-insensitive variant of the Na7H + exchanger found in rat hippocampal CA1 neurons. Therefore, in order to examine whether the effects of norepinephrine on resting pH ; were due to increased activity of the acid-extruding Na7H + exchanger, norepinephrine was applied in HEPES-buffered 52 medium in which N M D G + was substituted for external Na + . As shown in Fig. 14, exposure of the neuron to Na+-free, NMDG+-substituted HEPES buffered medium evoked a fall in pH, consequent upon blockade of Na7H + exchange (see also Fig. 5 A). In order to raise pH ; back to a more physiological level, 10 mM of the weak base trimethylamine (TMA) was then applied, in the continued absence of external Na + . Under these Na+-free conditions, 10 p M norepinephrine failed to evoke an increase in resting pHj. Similar results were obtained in a further 7 neurons tested. The mean rise in resting pHj evoked by 10 uM norepinephrine in H C 0 3 7 C 0 2 -buffered medium was compared to the average alkalinization of steady state pH ; induced by 10 p M norepinephrine during perfusion with HEPES-buffered medium. A n unpaired /-test indicated that the effects of 10 p M norepinephrine on steady state pHj in both types of media were not statistically different (P > 0.1). The average percentage increases in the rates of pH, recovery from an imposed acid load in the presence of 10 u M norepinephrine in both HC037C02-buffered and HEPES-buffered media were also compared and determined to be not statistically different by an unpaired /-test (P > 0.1). Since the effects of norepinephrine on resting pH ( and on the rates of pHj recovery from an imposed acid load were similar in both HC0 3/C0 2-buffered and HEPES-buffered media, the effects of norepinephrine are unlikely to reflect alterations in the activities of HC03"-dependent pH ; regulating mechanisms. Rather, the data indicate that in both HC0 37C0 2-buffered and HEPES-buffered media, the effects of norepinephrine on steady state pH; and on the rate of pHj recovery from an imposed acid load were most likely due to an enhancement of the activity of the acid extruding Na7H + exchanger. Thus, all 53 subsequent experiments were performed under HC03"-free, HEPES-buffered conditions to isolate the actions of norepinephrine on the activity of the Na7H + exchanger. i) Adrenergic receptor sub-types mediating the effects of norepinephrine on the activity of the Na7H+ exchanger The adrenergic receptor sub-types involved in norepinephrine-induced pHj modulation were determined by employing receptor-specific agonists and antagonists. In all experiments, P adrenergic receptor agonists were applied in the presence of a full a adrenergic receptor antagonist and vice versa, thus eliminating any possibility of cross reactivity by the adrenoceptor agonists. Thus, solutions containing isoproterenol, dobutamine or terbutaline also contained phentolamine, a non-specific a adrenergic receptor antagonist (Bylund et al, 1994), whereas media containing 6-fluoronorepinephrine also contained propranolol, a non-selective p-adrenergic receptor antagonist (Minneman et al., 1981; Bylund et al, 1994). Initial studies employed a non-selective ct-adrenergic receptor agonist, 6-fluoronorepinephrine (Cantacuzene et al, 1979), and a non-selective P-adrenergic receptor agonist, isoproterenol (Minneman et al, 1981; Bylund et al, 1994), both applied at a concentration of 10 uM. The a-adrenergic receptor agonist had no effect on steady state pH; in 5 neurons tested (Fig. 15A). In addition, 6-fluoronorepinephrine failed to alter the rate of pH ; recovery from an imposed intracellular acid load in 9 out of 9 neurons examined (Fig. 16A). In contrast, the p-adrenergic receptor agonist, isoproterenol, was able to mimic the effects of norepinephrine by increasing resting pHj by an average of 54 0.15 ± 0.03 pH units in 6 out of 7 neurons tested (Fig. 15B). The resting pH; of the remaining neuron did not change upon exposure to 10 p M isoproterenol. Isoproterenol also increased the rate at which pHj recovered from an imposed intracellular acid load by an average of 145% in 14 out of 16 neurons examined (Fig. 16B). In the two remaining cells, the rates of pH; recovery in the presence of 10 p M isoproterenol were increased by 5% and 31%, respectively. A comparison between the control rates of pH, recovery from an imposed acid load and the rates of pHj recovery of neurons that responded to 10 p M isoproterenol (n = 14) indicate that the ^-adrenergic receptor agonist was able to significantly increase the rates of pH ; recovery following an imposed intracellular acidification (P < 0.05 at all absolute levels of prf; Fig. 17A). In contrast, data from 9 neurons indicate that 10 p M 6-fluoronorepinephrine failed to alter significantly rates of pH; recovery from imposed acid loads (P > 0.1 at all absolute levels of plf; Fig. 17B). Selective agonists for both P T and P 2 adrenergic receptors, dobutamine and terbutaline (Minneman et al, 1981; Bylund et al, 1994), respectively, in the presence of a full a adrenoceptor antagonist, were able to mimic the actions of norepinephrine on pH;. In experiments involving dobutamine, ICI 118,551, a very selective p2-adrenergic receptor antagonist (O'Donnell and Wanstall, 1980; Fowler and O'Donnell, 1988; Booze et al, 1989) was also added to the perfusion medium together with phentolamine, to further increase the selectivity of the compound for p, adrenoceptors. In 9 out of 10 neurones examined, 1 p M dobutamine evoked an average rise in steady state pH ; of 0.18 ± 0.03 pH units (Fig. 18A). The resting pH; of the remaining neuron did not respond to an application of 1 p M dobutamine. Dobutamine 1 p M also increased the rates of pHi 55 recovery from an imposed intracellular acidification in 6 out of 7 neurons tested by an average of 134% as compared to the control rates of pH; recovery at the same absolute values of pHj (Fig. 18B). In the remaining neuron, dobutamine increased the rate of pHj recovery from an imposed acid load by an average of 16%. Terbutaline, a selective p2-adrenoceptor agonist, also evoked an increase in resting pHi. The effects of terbutaline on steady state pH; were concentration-dependent, as illustrated in Figure 19. Thus, 10 u M terbutaline evoked an average increase in resting pH, of 0.52 ± 0.04 pH units (n = 5) whereas 1.0 uM and 0.5 u M of the p2-adrenergic receptor agonist increased resting pHj by an average of 0.25 ± 0.02 pH units (n = 3), and 0.11 ± 0.03 pH units in = 3), respectively. In all neurones tested in =10), 1 u M terbutaline also increased the rates of pHi recovery from an imposed intracellular acid load by an average of 165% when compared to the control rates of pHi recovery at the same absolute values of pHi (Fig. 20). In Figure 21 A , a histogram is presented of the distribution of the resting pH; values of all neurons before exposure to norepinephrine or one of the p-adrenergic receptor agonists. The data were best fitted with the sum of two least squares Gaussian distributions with means at pIT 6.93 ± 0.03 and at pH ; 7.44 ± 0.02 (n = 50). Upon exposure to either norepinephrine, isoproterenol, dobutamine, or terbutaline, resting pH; alkalinized to a new steady state level. The resultant distribution of steady state pH; values was best fitted with a single least squares Gaussian distribution which was centered at pH : 7.54 ± 0.03 (Fig. 2IB). The results described above suggest that P-adrenoceptors mediate the effects of norepinephrine on both resting pH t and on the rate of pH; recovery following an imposed 56 intracellular acid load. In further support this possibility, 10 p M norepinephrine was combined with 20 p M propanolol, a full p-adrenergic receptor antagonist, and when applied had no effect on resting pH ; in the 4 hippocampal neurons tested (Fig. 22A). In addition, when the hippocampal neurons underwent an acid load and subsequent pHi recovery in the presence of 10 p M norepinephrine with 20 p M propranolol, in 7 neurons the rates of pH ; recovery were not altered, as compared to the control rates of recovery at the same absolute values of pHj (Fig. 22B). A comparison between the rates of pHj recovery under the influence of 10 p M norepinephrine (following pre-treatment with 20 p M propranolol) and the internal control rates of prl, recovery, at the same absolute values of pH i 5 is presented in Fig. 23 (n = 7). The results indicate that the non-specific P-adrenergic receptor antagonist blocked the previously documented norepinephrine-induced increases in the rates of pH, recovery after an imposed intracellular acid load, at all corresponding absolute values of pH;. The above results indicate that norepinephrine imparts its effects on resting pH, and on the rates of pH ; recovery following an imposed intracellular acidification via p-adrenergic receptors. C) Intracellular mechanisms of norepinephrine-induced actions on pH, i) Involvement of the Gs isoform of guanine nucleotide-binding regulatory proteins It is well known that norepinephrine interacts with P-adrenoceptors that are positively linked to adenylyl cyclase via the activation of an a-subunit of the G s isoform 57 of heterotrimeric G-proteins (Limbird, 1988). However, Barber and Ganz (1992) described a cholera toxin-insensitive G protein which mediated the effects of norepinephrine on the NHE-1 isoform of the Na7H + exchanger in canine enteric cells. Therefore, to investigate whether B-adrenoceptor-mediated modulation of pHj in hippocampal neurons was dependent upon the activation of G s , initial studies employed cultured post-natal hippocampal neurons pre-treated with 500 ng/ml cholera toxin, an irreversible activator of G s a , for 18 - 24 hours (Cassel and Pfeuffer, 1978; Limbird, 1988; Barber and Ganz, 1992). The use of cultured post-natal neurons was necessitated by the reported long incubation period (18 - 36 hours) required for activation of G s a by cholera toxin in other neuronal preparations (Thalmann, 1988; Harada et al, 1992; Ma et al, 1994). Cholera toxin acts by catalyzing the ADP-ribosylation of the GTP-binding protein, G s a , and uncouples it from both the B-adrenergic receptors and from the Py-dimer (Cassel and Pfeuffer, 1978; Gil l and Woolkalis, 1991; Wickman and Clapham, 1995). The bacterial enterotoxin, from Vibrio cholerae, also blocks the intrinsic GTPase activity of the a-subunit therefore leading to chronic stimulation of downstream effectors, in this case adenylyl cyclase (Cassel and Pfeuffer, 1978; Wickman and Clapham, 1995). If activation of a G s a subunit is involved in the norepinephrine-induced increase of resting pHi and the rate of pH, recovery following an imposed acid load, then prolonged pre-treatment with cholera toxin should persistently enhance the activity of the Na7H + exchanger and occlude further effects of norepinephrine on resting pH ; and on the rate of pH, recovery following an intracellular acid load. Therefore, resting pH, and the rate of pH ; recovery of these pre-treated neurons should not be affected by a subsequent 58 application of 10 p M norepinephrine. As anticipated, in pre-treated neurons, norepinephrine had no effect on resting pH, (n = 3; Fig. 24A) or on the rate of pHj recovery from an imposed intracellular acidification (n = 3; Fig. 25A). Untreated sister cultures were utilized as controls for these experiments. In cultured post-natal hippocampal neurons which had not been pre-treated with cholera toxin, 10 p M norepinephrine increased resting prf by an average of 0.24 ± 0.03 pH units (n = 3; Fig. 24B) and increased the rates of pHi recovery from an imposed intracellular acid load by an average of 116%, as compared to control rates of pH s recovery at the same absolute values of pH, (n = 3; Fig. 25B). Lahnsteiner and Hermann (1995) documented that, in the rat hippocampal slice preparation, an acute treatment with 40 nM cholera toxin for 20 minutes resulted in the activation of G s a . The effect of a similar acute treatment paradigm on both resting pH ; and on the rate of pH; recovery from an imposed intracellular acid load was therefore examined. Acute exposure of cultured post-natal hippocampal neurons to 50 n M cholera toxin evoked an average rise of 0.18 ± 0.03 pH units in steady state pH[ (n = 3; Fig. 26A). Subsequently, experiments using the acid load recovery paradigm were performed that also included a third acid load and subsequent pH, recovery under the influence of 10 p M norepinephrine (see Fig. 26B). Acute pre-treatment with 50 nM cholera toxin increased the rates of pH, recovery by an average of 179% as compared to the control rates of pH ; recovery at the same absolute values of pH, (n = 3; Fig 26B). Subsequent application of 10 p M norepinephrine failed to increase further the rates of pH ; recovery from an imposed acid load (under the influence of 10 p M norepinephrine, the rates of pH; 59 recovery were increased by an average of 206%, compared to the control rates of pHj recovery before the application of cholera toxin, at the same absolute values of pH f (n = 3; Fig. 26B)). Similar acute effects of cholera toxin were also seen in freshly isolated adult rat hippocampal CA1 neurons where, upon application of 50 nM cholera toxin, steady state pH; increased by an average of 0.15 + 0.02 pH units (n = 7/8; Fig. 27A). The resting pH; of the remaining neuron was not affected by the acute exposure to 50 nM cholera toxin. The rates of pH; recovery from an imposed intracellular acidification, in the presence of 50 n M cholera toxin, were increased by an average of 130% when compared to the control rates of pHi recovery at the same absolute values of p r i (n = 9/11; Fig. 27B). The average rates of pH ; recovery of the two remaining neurons were increased by only 3% and 16%, respectively. These results suggest that the G s class of G-protein a subunits mediates the effects of norepinephrine on the activity of the NaTFT exchanger in rat hippocampal neurons. ii) Involvement of second messengers: cAMP and calcium Occupation of P-adrenergic receptors by norepinephrine has been classically linked (via the GTP-G s a subunit) to activation of adenylyl cyclase and to an increased production of cyclic adenosine 3', 5'-monophosphate (cAMP). The next series of experiments determined that the activation of adenylyl cyclase and an increase in cytosolic [cAMP] mediated the effects of norepinephrine on steady state pH ; and on the rate of pHj recovery from an imposed acid load. 60 In the rat hippocampus norepinephrine, acting via both (3, and B2 adrenergic receptors, is able to generate cAMP after a short delay (-20 seconds after norepinephrine application) and the intracellular concentration of cAMP does not reach an asymptotic level until 5 minutes later (Dolphin et al, 1979; Segal et al, 1981a; Madison and Nicoll, 1986b). In acutely dissociated adult hippocampal CA1 neurons, forskolin was employed to enhance the activity of adenylyl cyclase (by reversibly binding with high affinity to the catalytic subunit of the enzyme (Seamon and Daly, 1981; Daly et al, 1982)) and thereby increase cytosolic [cAMP]. 20-25 uM of the diterpene, in the absence of norepinephrine, was able to mimic the actions of the catecholamine on pHj. Forskolin 20 u M increased cytosolic pH by an average of 0.19 + 0.03 pH units (n = 9/10; Fig. 28A). The steady state pHi of the remaining neuron was unaffected by an application of 20 uM forskolin. The rates of pHj recovery from an intracellular acid load under the influence of 25 u M forskolin were greater than those observed under control conditions by an average of 169%, at corresponding absolute values of pH; (n = 5; Fig. 28B). Mammalian adenylyl cyclases are directly inhibited by analogs of adenosine such as 2',5'-dideoxyadenosine (Chijiwa et al, 1990; Johnson and Shoshani, 1990; Johnson and Shoshani, 1994). The actions of norepinephrine on steady state pHj were blocked by pre-treatment with 100 u M 2',5'-dideoxyadenosine (n = 6; Fig. 29A). Following pre-treatment with 100 u M 2',5'-dideoxyadenosine, the rates of pHi recovery from an imposed acid load in the presence of 10 uM norepinephrine were comparable to those imposed under control conditions, when compared at the same absolute values of pH; (n = 7; Fig. 29B). A naturally occurring inactive analog of forskolin, l',9'-dideoxyforskolin (Laurenza 61 et al, 1989), did riot increase resting pH ; (n = 5; Fig. 30A) or the rate of pH, recovery from an imposed acid load (n = 7; Fig. 30B). Cyclic nucleotide phosphodiesterases catabolize cyclic nucleotides, thereby regulating their activity. Nonspecific phosphodiesterase inhibitors such as 3-isobutyl-l-methylxanthine (IBMX) block, amongst others, cAMP-dependent phophodiesterases, thus increasing the intracellular concentration of cAMP (Smellie et al, 1979; Wu et al, 1982). Exposure of acutely dissociated rat hippocampal CA1 neurons to 200 p M I B M X caused an average increase in resting pH; of 0.16 ± 0.01 pH units, measured at 10 to 15 minutes following the start of exposure to the phosphodiesterase inhibitor (n - 10/11; Fig. 31). A possible reason for the relatively long delay in the effects of 200 p M I B M X on resting pH, may be due to the low membrane permeability of the methylxanthine (Thompson, 1991). The resting pH, of the remaining neuron was unaffected by application of 200 p M IBMX. Taken together, the above results indicate that the p-adrenergic receptor-mediated effects of norepinephrine on both steady state pH; and on the rate of pH ; recovery from an imposed intracellular acid load are mediated by a signal transduction pathway involving activation of adenylyl cyclase and the subsequent production of cAMP. The results also indicate that, in the absence of norepinephrine, activation of this signal transduction pathway can modulate the activity of the Na7H + exchanger in rat hippocampal neurons. To investigate whether the effects of norepinephrine on pIF might be secondary to changes in [Ca2+]j, experiments were performed during perfusion with Ca2+-free, HEPES-buffered medium (Table 1, solution 3) containing 200 p M ethylene glycol-bis(P-62 aminoethyl ether)-./V, N, N1, ./V-tetraacetic acid (EGTA). Exposure to Ca2+-free medium per se caused an increase in resting pH ; of variable magnitude (Fig. 32) and the subsequent application of 10 p M norepinephrine evoked a further rise in resting pHi of 0.18 ± 0.01 pH units (n = 10/13; Fig. 32A). The steady state pHiS of the remaining neurons were not affected by 10 p M norepinephrine under Ca2+-free conditions. Norepinephrine 10 p M also increased the rates of pHi recovery from an imposed acid load by an average of 145% when compared to the control rates of pH; recovery at the same absolute values of pHj (n = 6/7; Fig. 32B). In the remaining neuron, under Ca2+-free conditions, 10 p M norepinephrine increased the rate of pH; recovery from an imposed acid load by an average of 8%, when compared to the control rates of pHj recovery at the same absolute values of plf. The actions of isoproterenol were also examined under zero external Ca 2 + conditions. The P-adrenergic receptor agonist (tested at 10 p M in the presence of 10 p M phentolamine) initiated an average alkalinization of steady state pH; of 0.18 ± 0.02 pH units (n = 4; Fig. 33A). Under Ca2+-free conditions, 10 p M isoproterenol with 10 p M phentolamine also increased the rates of pHj recovery from an acid load by an average of 160% when compared to the control rates of pH; recovery at the same absolute values of pH ; (n - 6; Fig. 33B). The results indicate that the effects of P~ adrenergic receptor agonists on both resting pH, and the rate of pH; recovery from an imposed intracellular acid load are not dependent upon Ca 2 + entry into the neuron. It remains possible that the effects of norepinephrine and P-adrenergic receptor agonists on steady state pH, and on the rate of pH ; recovery from an imposed acid load may be secondary to a rise in [Ca2+]i consequent upon the release of Ca 2 + from 63 intracellular stores. Therefore, experiments were performed in both cultured post-natal and freshly isolated adult rat hippocampal neurons loaded with the fluorescent Ca 2 + indicator, fura-2 (Grynkiewicz et al, 1985), to examine directly the effects of 10 u M isoproterenol with 10 uM phentolamine on [Ca2+]j. In cultured post-natal hippocampal neurons perfused with Ca2+-free medium, 10 uM isoproterenol applied in the presence of 10 u M phentolamine had no effect on the mean BI334/BIJS0 (background-corrected I33JI3$0) value and, thus, [Ca2+] ; (n = 4; Fig. 34A). In 5 neurons acutely isolated from the CA1 region of an adult rat hippocampus, the mean BI33JBI3i0 value was unaffected by 10 u M isoproterenol with 10 u M phentolamine (Fig. 34B). Taken together, these results suggest that both norepinephrine and isoproterenol enhance the activity of the Na7H + exchanger in a Ca2+-independent manner. iii) Involvement of protein kinase A The norepinephrine-induced increase of steady state pHj and of the rate of pH; recovery after an imposed intracellular acidification also involved the activation of the cAMP-dependent protein kinase (protein kinase A ; PKA) . The involvement of P K A was determined using a very selective activator of P K A , the Sp-diastereomer of c A M P (Sp-cAMPS), and two selective P K A inhibitors, the Rp-diastereomer of cAMP (Rp-cAMPS) and A^-[2-(jD-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide*2HCl (H-89; Parker-Bothelo et al, 1988; Hidaka and Kobayashi, 1992; Yuan and Bers, 1995). In acutely dissociated adult rat hippocampal CA1 neurons, 25 u M Sp-cAMPS evoked a mean rise in resting pHj of 0.19 ± 0.01 pH units (n= 11/13; Fig. 35A). The resting pH, of 64 the remaining two neurons was unaffected by application of 25 p M Sp-cAMPS. In 9 out of 10 adult hippocampal CA1 neurons, rates of pH< recovery from an imposed intracellular acid load in the presence of 25 p M Sp-cAMPS were increased by an average of 168% when compared to the control rates of recovery at the same absolute values of pHj (Fig. 35B). In the remaining neuron, 25 p M Sp-cAMPS increased the rates of pH; recovery from an imposed acid load by an average of 33% when compared to the control rates of pH; recovery at the same absolute values of pH ; . Both Rp-cAMPS (50 pM) and H-89 (10 pM) blocked the effects of norepinephrine on resting pHj (n = 5, Fig. 36A and n = 10, Fig. 37A, respectively). The effect of 10 p M norepinephrine on the rate of pH; recovery from an imposed acid load was also blocked by pre-treatment with either 50 p M Rp-cAMPS or 10 p M H-89 (« = 4, Fig. 36B and n = 5, Fig. 37B, respectively). Thus, the effects of norepinephrine on steady state pHj and on the rate of pH ; recovery after an imposed intracellular acidification likely involves the phosphorylation of the Na7H + exchanger by P K A and a subsequent increase in the rate of activity of the exchanger. In summary, during perfusion with HEPES-buffered medium, norepinephrine increased resting pH< and the rate of pH; recovery from an imposed intracellular acidification by enhancing the operation of the main acid extrusion mechanism present in hippocampal neurons, the Na7H + exchanger. This calcium-independent alteration in the activity of the Na7H + exchanger was P-adrenergic receptor-mediated and also involved the activation of G s a , adenylyl cyclase, and protein kinase A. 65 Figure 3. Distributions of steady state pHj values for all neurons during perfusion with HEPES-buffered and HC037C02-buffered media A . The distribution of the resting pH f values for 439 adult rat hippocampal CA1 neurons during perfusion with HEPES-buffered saline (bin width = 0.05 pH units). The data were best fitted with a bimodal Gaussian distribution with means at pHj 6.91 ± 0.01 and at pHi 7.43 ± 0.01. B. The distribution of the resting pH; values for 55 neurons during perfusion with HC037C02-buffered saline (bin width = 0.05 pH units) was fitted with a Gaussian distribution centered at pH ; 7.20 ± 0.03. 66 67 Figure 4. Effects of EIPA on steady state pH( and on the recovery of pHj from an intracellular acid load in HEPES-buffered saline A single adult rat hippocampal CA1 neuron had an initial resting pHi of ~ 7.02. 50 p M EIPA failed to block the recovery of pH ; from an intracellular acid load imposed by an N H 4 + prepulse and, in a subsequent application, did not affect steady state pH;. r 68 69 Figure 5. Effects on steady state pH, of perfusion with Na+-free, HEPES-buffered media A . The application of HEPES-buffered saline with external Na + replaced with N M D G + , at a constant pH 0 , decreased pH; by ~ 0.25 pH units. pH ; recovered when external Na + was re-introduced. B. During exposure to a HEPES-buffered perfusate in which Na + was replaced by L i + , resting pH; transiently decreased by ~ 0.46 pH units. However, pH; recovered fully in the continued absence of external Na + . Each trace represents data obtained from different neurons. 70 B. 8 7.6 X 7.4 a. 7.2 0 [Na + ] o /NMDG 0 [Na + ] 0 /Li + 7 6.8 12 16 Time (min) 20 24 71 Figure 6. Effects of perfusion with Na+-free, HEPES-buffered media on recovery of pH, from an imposed intracellular acidification A . Following exposure to an NH4+-containing HEPES-buffered saline, cytosolic pH acidified beyond the previous resting level. At the point of maximum acidification, external Na + was replaced with N M D G + . This maneuver abolished any recovery from the imposed intracellular acid load until external Na + was re-introduced. B. When extracellular Na + was replaced by L i + , at the point of maximum acidification following an N H 4 + prepulse, pH ; recovered fully from an imposed acid load in the continued absence of external Na + . Each trace represents data collected from different neurons. 73 Figure 7. The effects of the transition from HEPES-buffered saline to H C 0 3 7 C 0 2 -buffered medium on resting pH ; The magnitude of the increase in resting pHj following the transition from a HEPES-buffered perfusate to a HC037C02-buffered medium, at a constant pH 0 , depended on the initial pH ; in HEPES-buffered medium. A . During perfusion with HC0 37C0 2-free, HEPES-buffered medium, this hippocampal neuron had a resting pH; of 6.86. Upon exposure to HC03"-containing saline (at a constant pH0), pH ; increased by ~ 0.34 pH units. When external HCOy was removed, pH; returned to previous resting levels. B . From an initial pH ; of 7.23, the neuron experienced a transient acidification followed by a very small increase in resting pHj upon exposure to a HC037C02-buffered perfusate. C. Pooled data from 20 neurons illustrates that the increase in resting pH, upon the addition of HC0 3 " to the perfusate was dependent on the initial steady state pH; in HEPES-buffered medium. The continuous line represents a linear least-squares best fit (r^= 0.83) for the initial pH ; (in HC0 37C0 2-free, HEPES-buffered medium) against the change in pH, (upon exposure to HC03"-containing medium, at a constant pH 0). 74 • 75 Figure 8. Effects of DIDS and 0 [CI]0 on steady state pH ; in the presence of HCGy A. During perfusion with a HC03"-free, HEPES-buffered medium, resting pHj was ~ 7.16. Upon exposure to a HC037C02-buffered medium containing 300 u M DIDS (at a constant pH 0), pH ; fell slightly (compare with Fig. 7A). Upon removal of DIDS, pH ; rose to a new steady state value of - 7.42. B. In an experiment conducted in the presence of HCGy, the removal of external CI", at a constant pH Q , caused an increase of resting pH;. pH; returned to normal levels upon the re-introduction of external CI". A subsequent withdrawal of external CI", on this occasion in the presence of 300 u M DIDS, markedly attenuated the 0[Cl"]o-evoked rise in resting pH,. Each trace represents data collected from a different neuron. 76 77 Figure 9. Effects of 0 [Na+]0 on resting pH; and on the recovery of pH, from an imposed intracellular acidification in the presence of HC03" A. Extracellular Na + was substituted with N M D G + and upon application caused steady state pH; to decrease by ~ 0.54 pH units. Upon the re-introduction of external Na + , pH; recovered to previous steady state levels. B. When external Na + was removed at the peak intracellular acidification following an N H 4 + prepulse, recovery of pHj to previous steady state pHj was blocked until the re-introduction of extracellular Na + . Each trace represents data obtained from a different neuron. 79 Figure 10. Effects of norepinephrine on steady state pH; and on the rate of pHj recovery from an imposed cytosolic acidification, in the presence of HC03" A. From an initial resting pH; of ~ 7.05, a ten minute application of 10 u M norepinephrine (NE) increased resting pH ; by ~ 0.25 pH units. B. An initial acid load was performed under control conditions. Following the recovery of pH; back to the control resting level, a second acid load was performed, this time under the influence of 10 u M norepinephrine (NE). In the presence of 10 u M norepinephrine (NE), the rate of pH; recovery increased by an average of 164% as compared to the rate of pH; recovery following the first acid load. Note also that, in the presence of 10 uM norepinephrine (NE), pHi recovered to a higher resting level than that observed in the absence of norepinephrine (NE). Each trace represents data obtained from a different neuron. 81 Figure 11. Effects of norepinephrine on steady state pHj and on the rates of pH; recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium A . From an initial resting pHj of ~ 7.55, an application of 10 p M norepinephrine (NE) increased steady state pH i 5 after a short delay of ~3 minutes, by ~ 0.35 pH units. B. An initial acid load maneuver was performed under control conditions. Upon full recovery of resting pH ; , a second acid load was performed in the presence of 10 p M norepinephrine (NE). 10 p M norepinephrine (NE) increased the rate of pH; recovery from an imposed intracellular acidification by an average of 193%, as compared to the control rate of recovery from the initial acid load. Each trace represents data collected from a different neuron. 82 83 Figure 12. Long-term effect of norepinephrine on steady state pH; during perfusion with HEPES-buffered medium A . Upon exposure to 10 p M norepinephrine (NE), the initial resting pHj value of -7.40 began to rise after a delay of ~ 3 minutes and eventually reached a maximum level of pH; ~ 7.61. Prolonged washout of the catecholamine (~ 30 minutes) failed to return the alkalinized level of resting pHj to the level observed prior to the application of norepinephrine (NE). B. The stability of the corresponding values of BI452 (background-corrected values of 7452), during the experiment depicted in Fig. 12A, indicate that the membrane integrity of the neuron was not comprised during the long-term recording (see Materials and Methods). Both traces were obtained simultaneously from the same neuron. 8 4 85 Figure 13. Comparison of the control rates of pHj recovery to rates of pH; recovery under the influence of norepinephrine during perfusion with HEPES-buffered medium Plotted are average rates of pH ; recoveries from acid loads in the presence (•) and absence (A) of 10 uM norepinephrine, at pH; values shown on the abscissa. Rates were evaluated at 0.05 unit intervals of pH, and error bars represent S.E.M. Continuous lines represent the least squares linear regression fits to the data points indicated for each experimental condition. Data were obtained from 15 paired experiments of the type shown in Fig. 1 IB. At each absolute level of pH i 3 norepinephrine 10 u M significantly increased the rates of pHj recovery following an imposed intracellular acid load as compared to the rates of pHj recovery imposed under control conditions (P<0.05 for each absolute value of pHj. 0.020 -, 0.016 J 0.012 J •a 0.008 J 0.004 J 0.000 6. 87 Figure 14. Effect of norepinephrine on steady state pH; during perfusion with a Na+-free, HEPES-buffered medium At a constant pH 0 , the hippocampal CA1 neuron was exposed to a Na+-free, HEPES-buffered medium (replaced with NMDG + ) which caused resting pH; to decrease by ~ 0.39 pH units. Subsequent application of the weak base trimethylamine (TMA) raised pH; to a more physiologically relevant level (~ pH; 7.02). A subsequent application of 10 u M norepinephrine (NE) failed to evoke a change in steady state pH ; (compare with Fig. 11 A). 88 X 0< 7.6 7.4 -7.2 -7 6.8 6.6 6.4 6.2 0 0 [ N a + ] 0 / N M D G + l O m M T M A 10 15 20 Time (min) 10 uM N E 25 30 89 Figure 15. Effects of 6-fluoronorepinephrine and isoproterenol on steady state pH| during perfusion with HEPES-buffered medium A. The adult hippocampal neuron was perfused with HEPES-buffered medium containing 10 uM 6-fluoronorepinephrine (6-FNE) with 10 u M propranolol (PRO) for ~ 10 minutes. Resting pH; was not affected. B. Upon application of a HEPES-buffered medium containing 10 uM isoproterenol (ISO) and 10 u M phentolamine (PHE), resting pHi increased by ~ 0.17 pH units after a delay of ~3 minutes and reached a maximum level within ~ 9 minutes of exposure to the B-adrenergic receptor agonist. Each trace represents data recorded from a different neuron. 90 91 Figure 16. Effects of 6-fluoronorepinephrine and isoproterenol on rates of pH; recovery from an imposed intracellular acidification in HEPES-buffered medium A . A n initial acid load maneuver was performed under control conditions and, following recovery of pH ;, a second acid load and subsequent recovery of pH; was conducted under the influence of 10 p M 6-fluoronorepinephrine (6-FNE) and 10 p M propranolol (PRO). The rates of pHj recovery under the influence of 10 p M 6-fluoronorepinephrine (6-FNE) and 10 p M propranolol (PRO) were, on average, 37% slower than the internal control rates of recovery when compared at all corresponding absolute values of pHj. B. The rates of pH; recovery of the initial acid load served as a control for comparison to the second acid load and subsequent pHj recovery performed in the presence of 10 p M isoproterenol (ISO) and 10 p M phentolamine (PHE). For this particular neuron, the rates of pHi recovery under the influence of 10 p M isoproterenol (ISO) and 10 p M phentolamine (PHE) were, on average, 143% greater than the rates of pH; recovery imposed under control conditions when compared at corresponding absolute values of pHj. Each trace represents data recorded from a different neuron. 9 2 9 3 Figure 17. Comparison of the rates of pH; recovery under the influence of either isoproterenol or 6-fluoronorepinephrine and control rates of pHi recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium A . Plotted are the mean rates of pHj recoveries from intracellular acid loads conducted under the influence of 10 u M isoproterenol with 10 u M phentolamine (•) and the corresponding rates of pHj recovery performed under control conditions (A), at levels of pH; denoted on the x-axis. The rates of pHj recovery were evaluated at 0.05 unit intervals of pH, and error bars represent S.E.M. Continuous lines represent the least squares linear regression fits to the data points indicated for each experimental condition. Data were obtained from 14 paired experiments of the type shown in Fig. 16B. At each absolute level of pH i ; isoproterenol 10 uM significantly increased the rates of pHj recovery following an intracellular acid load as compared to the control rates of pH ; recovery (PO.05 at each absolute value of pH ;). B. The average rates of pH; recovery from an intracellular acid load in the presence of 10 uM 6-fluoronorepinephrine with 10 u M propranolol (•) were compared to the average control rates of pHj recovery (A) at values of pHi denoted on the x-axis. Rates of pH-, recovery were evaluated at 0.05 unit intervals of pHj and error bars represent S.E.M. Continuous lines denote the least squares linear regression fits to the data points for each experimental condition. Data were obtained from 9 paired experiments of the type shown in Fig. 16A. The rates of pHj recovery in the presence of 10 u M 6-fluoronorepinephrine with 10 u M propranolol were not significantly different at any level of pHj when compared to the corresponding control rates of pH> recovery (P > 0.1 at each absolute level of pH;). 94 A . 0.015 - . 0.012 - . 95 Figure 18. Effects of dobutamine on steady state pHj and on the rate of pH( recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium A . The hippocampal neuron was pre-treated for 5 minutes with HEPES-buffered medium containing 10 p M ICI 118,551, a selective pVadrenergic receptor antagonist. The neuron was then exposed to 1 p M dobutamine (DBT) with 1 p M phentolamine (PHE). After ~ 5 minutes, resting pH; began to rise and attained a maximum level after ~ 14 minutes of exposure (ApH; = 0.18 pH units). B. The neuron underwent an initial acid load and subsequent pH s recovery under control conditions. After pH ; had fully recovered, the neuron was exposed to 1 p M dobutamine (DBT), l p M phentolamine (PHE) and 10 p M ICI 118,551 at the beginning of the second acid load. The rates of pH; recovery from the resultant intracellular acidification were increased by an average of 127% as compared to the internal control rates of pH; recovery at all corresponding values of pH,. Each trace represents data collected from different neurons. 96 A . 7.2 -, lOuMICT 118,551 1 uM DBT + 1 uM PHE 7.1 X 6.9 Time (min) 97 Figure 19. Concentration dependent effects of terbutaline on steady state pHi during perfusion with HEPES-buffered saline A . After 4 minutes of exposure to 0.5 p M terbutaline (TRB) with 0.5 p M phentolamine (PHE), resting pHj of 7.41 began to rise and reached a maximum level of pH; 7.53. B. The initial resting pH ; of 6.89 was raised to pH, 7.28 during an 11 minute application of the [^-adrenergic receptor agonist (1 pM) with the non-selective a-adrenoceptor antagonist (1 pM). C. In this neuron, a 9 minute exposure to 10 p M terbutaline (TRB) with 10 p M phentolamine (PHE) caused resting pH; to rise by ~ 0.51 pH units. Each trace represents data obtained from different adult hippocampal CA1 neurons. 98 A . 7.7 7.6 a 7.5 B. C. 7.4 7.3 7.6 7.4 ffi 7.2 a. 7 6.8 7.8 7.6 -1 7.4 7.2 7 6.8 0 0.5 uM TRB + 1 u M PHE 2 4 6 8 10 12 14 Time (min) 1 uM TRB + 1 u M PHE 5 10 Time (min) 1 0 u M T R B + l uM PHE 4 8 12 Time (min) 15 16 99 Figure 20. Effect of terbutaline on the rates of recovery of pHj from an imposed intracellular acid load during perfusion with HEPES-buffered medium In this trace, the initial acid load served as a control for comparison to the second acid load and subsequent recovery of pHj performed under the influence of 1 uM terbutaline (TRB) with 1 u M phentolamine (PHE). The rates of pH> recovery, in the presence of 1 u M terbutaline (TRB) with 1 uM phentolamine (PHE), were significantly increased by an average of 159%, when compared to the internal control rates of recovery at the same absolute levels of pH;. Also shown is a one-point calibration during perfusion with a high [K +], HEPES buffered medium containing 10 uM nigericin at pH 7.00. 100 Time (min) 101 Figure 21. Effect of norepinephrine and P-adrenergic receptor agonists on the distribution of the steady state pH, values of adult rat hippocampal CA1 neurons during perfusion with HEPES-buffered media A . The distribution (bin width = 0.05 pH units) of the resting pH; values of adult hippocampal CA1 neurons, before exposure to norepinephrine or P-adrenergic receptor agonists (n - 50), was best fitted with the sum of two Gaussian distributions with means at pHi 6.93 ± 0.03 and at pH, 7.44 ± 0.02. B. After exposure to either norepinephrine, isoproterenol, dobutamine, or terbutaline, steady state pHi increased to a new resting value (n = 50). The distribution of alkalinized values of steady state pH; was best fitted with a single Gaussian distribution centered at pH; 7.54 ± 0.03 (bin width = 0.05 pH units). 102 103 Figure 22. Effects of 10 uM norepinephrine with 20 uM propranolol on steady state pHj and on the rate of pH ; recovery after an imposed intracellular acid load during perfusion with HEPES-buffered medium A. The hippocampal CA1 neuron had an initial resting pH : of ~ 7.39. 20 p M propranolol (PRO) was applied ~ 9 minutes before the neuron was exposed to 10 p M norepinephrine (NE). In the continued presence of 20 p M propranolol (PRO), 10 p M norepinephrine (NE) had no effect on resting pHj. Also shown is a one-point calibration during perfusion with a high [K +], HEPES buffered medium containing 10 p M nigericin at pH 7.00. B. An initial acid load and subsequent recovery of pHj was performed under control conditions. After full recovery of pH i ; 20 p M propranolol (PRO) was applied 3 minutes preceding the start of a second acid load. The second acid load and subsequent pH; recovery was conducted in the presence of both 10 p M norepinephrine (NE) and 20 p M propranolol (PRO). The rates of pH; recovery under the influence of 10 p M norepinephrine (NE) with 20 p M propranolol (PRO) were comparable to the internal control rates of pHj recovery at all corresponding levels of pHj. Each trace represents data obtained from different neurons. 104 A . 7.9 7.7 7.5 7.3 7.1 20 uM PRO 10 uM nigericin at pH 7.00 10 uM N E 6.9 10 15 20 25 Time (min) 30 35 40 B. 8.4 * 7-6-1 7.2 6.8 N H , + 20 u M PRO 10 u M N E N H / 3 6 9 12 15 18 21 Time (min) 105 Figure 23. Comparison between control rates of pH ( recovery and rates of pH t recovery under the influence of 10 uM norepinephrine with 20 uM propranolol during perfusion with HEPES-buffered medium The graph illustrates a comparison between control rates of pH ; recovery (A) and rates of pH; recovery in the presence of 10 uM norepinephrine following pre-treatment with 20 u M propranolol (•) at pH, levels denoted by the x-axis. The rates of p H recovery were evaluated at 0.05 unit intervals of pH, and error bars represent S.E.M. Continuous lines represent the least squares linear regression fits to the data points indicated for each experimental condition. Data were obtained from 7 paired experiments of the type shown in Fig. 22B. The rates of pH ; recovery after an imposed acid load under the influence of 10 u M norepinephrine following pre-treatment with 20 p M propranolol were not significantly different from control rates of recovery (P > 0.1 at each absolute level of pH ;). Compare with Figure 13. 106 0.010 - , 107 Figure 24. Effect of a pre-treatment with cholera toxin on the norepinephrine-induced increase in steady state pH, during perfusion with HEPES-buffered medium A . Cultured post-natal hippocampal neurons were pre-treated with 500 ng/ml cholera toxin for ~ 18 hours. The neurons, with an initial mean steady state pHj of ~ 7.38, were exposed to 10 p M norepinephrine (NE) for ~ 11 minutes and the resting pH; was not affected. The trace represents the mean of data simultaneously recorded from 21 neurons on a single coverslip. Also shown is a one-point calibration conducted with a high [K + ] , HEPES-buffered medium containing 10 pM nigericin at pH 7.00. B. Sister cultures of post-natal neurons were not pre-treated with cholera toxin. Upon exposure to 10 p M norepinephrine (NE), resting pH; increased by ~ 0.29 pH units after a delay of ~ 3 minutes and reached a maximum level after - 1 0 minutes. The trace represents the mean of data collected from 18 neurons on a single coverslip. Time (min) 109 Figure 25. Effect of a pre-treatment with cholera toxin on the norepinephrine-induced increase in the rate of pHj recovery from an imposed acid load during perfusion with HEPES-buffered medium A . Cultured post-natal hippocampal neurons were incubated in 500 ng/ml cholera toxin for - 21 hours. An initial acid load was performed under control conditions. Following the recovery of pHjback to control levels, a second acid load and subsequent pH ; recovery was conducted in the presence of 10 uM norepinephrine (NE). The rates of pH ; recovery from an acid load under the influence of 10 u M norepinephrine (NE) were comparable to the control rates of pH ; recovery at all corresponding levels of pH ; . The trace represents the mean of data recorded from 16 neurons on a single coverslip. B. Two acid loads were performed on sister cultures of post-natal neurons not pre-treated with cholera toxin. The initial acid load was imposed under control conditions. Following recovery of pH i 5 a second acid load and subsequent pH, recovery was performed in the presence of 10 u M norepinephrine (NE). The rates of pH : recovery under the influence of 10 u M norepinephrine (NE) were increased by an average of 132% when compared to the control rates of pH ( recovery at the same absolute levels of pH ; . The trace depicts the mean of data collected from 21 neurons on a single coverslip. Time (min) I l l Figure 26. Effects of an acute exposure to cholera toxin on resting pH; and a comparison of the rates of pH; recovery under the influence of cholera toxin and norepinephrine in cultured post-natal hippocampal neurons during perfusion with HEPES-buffered medium A . Cultured post-natal hippocampal CA1 neurons had an average resting pHj of ~ 7.03. Upon application of 50 nM cholera toxin (CTX), resting pH, began to increase after ~ 3 minutes and reached a maximum of phi 7.24 after - 3 0 minutes of exposure. This trace represents the mean of data collected from 10 neurons on a single coverslip. B. A series of three acid loads and subsequent pHj recoveries were performed. The rates of pH, recovery from the initial acid load served as the controls for comparison with the rates of pHj recovery from the second and third acid loads under the influence of 50 n M cholera toxin (CTX) and 10 p M norepinephrine (NE), respectively. Following recovery of pHi from the initial acid load, cultured hippocampal neurons were exposed to 50 n M cholera toxin (CTX) for - 14 minutes before the second acid load was initiated. Cholera toxin (CTX) increased the rates of pHi recovery by an average of 179% as compared to the control rates of pH[ recovery. 10 p M norepinephrine (NE) did not further augment the rates of pHj recovery from the third acid load as compared to the rates of pH ( recovery from the second acid load. The trace represents the mean of data collected from 19 neurons on a single coverslip. 112 A . 7.5 7.4 7.3 X 7.2 -j 7.1 6.9 -, r 0 5 50 nM C T X 10 uM nigericin — at pH 7.00 10 15 20 25 30 35 40 45 Time (min) 50 nM C T X 0 4 8 12 16 20 24 28 32 36 40 44 Time (min) 113 Figure 27. Effects of cholera toxin on steady state pHj and on the recovery of pHj from an imposed intracellular acidification in freshly isolated adult hippocampal CA1 neurons perfused with HEPES-buffered medium A . The adult hippocampal neuron had an initial resting pHj of ~ 7.30. Upon exposure to 50 n M cholera toxin (CTX), resting pH; increased by 0.17 pH units after a delay of ~ 4 minutes and reached the new steady state level of pH> after 14 minutes of exposure to the G s a activator. B. After the initial acid load and subsequent recovery of pH i 5 exposure to 50 n M cholera toxin (CTX) began ~ 7 minutes before the second acid load. The rates of pH, recovery under the influence of 50 nM cholera toxin (CTX) were increased by an average of 217% as compared to the control rates of pH ; recovery, at all corresponding absolute values of pH^ Each trace represents data collected from different neurons. 115 Figure 28. Effects of forskolin on steady state pH; and on the rates of pH ; recovery from an imposed intracellular acidification during perfusion with HEPES-buffered medium A . A n adult hippocampal CA1 neuron was exposed to 20 u M forskolin. This maneuver caused steady state pHj to increase by ~ 0.25 pH units, the rise beginning after a delay of ~ 3 minutes. B. A n initial acid load was performed under control conditions. Following recovery of pH i 5 a second acid load and subsequent recovery of pHj was conducted under the influence of 25 u M forskolin. The rates at which pH ; recovered after the second intracellular acid load, in the presence of 25 u M forskolin, were increased by an average of 180% when compared to the control rates of pH ; recovery at the same absolute values of pH,. Each trace represents data recorded from different neurons. 117 Figure 29. Effects of 2',5'-dideoxyadenosine on norepinephrine-induced effects on steady state pHj and on the rates of pH, recovery from an imposed acid load during perfusion with HEPES-buffered medium A . A n adult hippocampal CA1 neuron, with an initial resting pH; of - 7.51, was exposed to 100 p M 2',5'-dideoxyadenosine for 12 minutes prior to the application of 10 p M norepinephrine (NE). Under these conditions, 10 p M norepinephrine (NE) had no effect on resting pH ; . B. An initial acid load was performed under control conditions. Following recovery of pH ; , the neuron was perfused with HEPES-buffered saline containing 100 p M 2',5'-dideoxyadenosine for 5 minutes prior to the start of the second acid load. The subsequent recovery of pHj took place in the presence of 10 .pM norepinephrine (NE) and 100 p M 2',5'-dideoxyadenosine. In the continued presence of 100 p M 2',5'-dideoxyadenosine, 10 p M norepinephrine (NE) had no effect on the rates of pHj recovery. Each trace represents data collected from different neurons. 118 A . 7.8 7.7 7.6 ffi 7.5 7.4 7.3 100 u M 2',5'-dideoxyadenosine l O u M N E 7.2 12 16 Time (min) 20 24 28 B. 8.4 8.2 8 -I 7.8 7.6 7.4 7.2 -I 7 N H , + 100 uM 2',5'-dideoxyadenosine 10 u M N E N H , 0 12 15 18 21 24 27 Time (min) 119 Figure 30. Effects of l',9'-dideoxyforskoIin on steady state pH, and on the rates of pH, recovery from an imposed acid load during perfusion with HEPES-buffered medium A . A n adult hippocampal neuron was exposed to an ~ 11 minute application of 25 p M r,9'-dideoxyforskolin, during which steady state pH; was not affected (compare with Fig. 28A). B. An initial acid load and subsequent recovery of pHj was conducted under control conditions. A second acid load was then performed in the presence of 25 p M r,9'-dideoxyforskolin. 25 p M l',9'-dideoxyforskolin did not increase the rates of pH; recovery from an intracellular acid load when compared to the rates of pH; recovery performed under the control conditions (compare with Fig. 28B). Each trace represents data obtained from different neurons. 120 Time (min) 121 Figure 31. Effect of IBMX on steady state pH, during perfusion with HEPES-buffered medium A n adult hippocampal neuron, with an initial resting level of pHj ~ 7.33, was exposed to 200 u M I B M X . After ~ 23 minutes of exposure to the phosphodiesterase inhibitor, resting pH ; began to rise and reached a maximum level of pHj 7.45 after ~ 45 minutes of exposure to 200 u M IBMX. 122 Time (min) 123 Figure 32. Effects of 0 [Ca2+]0 on norepinephrine-induced changes in steady state pH, and in the rates of pH; recovery from an imposed acid load during perfusion with HEPES-buffered medium A . A n adult hippocampal neuron was perfused with Ca2+-free, HEPES-buffered saline containing 200 p M E G T A and resting pHj increased by ~ 0.12 pH units. When pH; stabilized at a new steady state level, an application of 10 p M norepinephrine (NE) increased steady state pH ; by an additional 0.11 pH units in the continued absence of external Ca 2 + . B. Two acid loads were performed during perfusion with Ca2+-free, HEPES-buffered medium. The initial acid load was imposed under control conditions while the second acid load and subsequent recovery of pH ; was performed in the presence of 10 p M norepinephrine (NE). The rates of pH; recovery from the intracellular acid load were increased by an average of 228% under the influence of 10 p M norepinephrine (NE) when compared to the control rates of pH ; recovery at all corresponding levels of pH ; . Each trace represents data recorded from different neurons. 124 A . 7.6 7.5 7.4 0 [Ca 2 +] 0 + 200 uM E G T A 10 u M N E K 7.3 7.2 7.1 ~i 1 10 15 20 25 30 35 40 Time (min) B. 7.9 0 [Ca 2 +] 0 + 200 uM E G T A 7.7 X 7.5 7.3 10 u M N E 7.1 0 3 6 9 12 15 18 21 24 27 Time (min) 125 Figure 33. Effects of perfusion with a Ca2+-free, HEPES-buffered medium on isoproterenol-induced changes in steady state pHj and in the rates of pH; recovery from an imposed acid load A . The acutely dissociated hippocampal CA1 neuron was perfused with a Ca2+-free, HEPES-buffered medium, containing 200 p M EGTA, for ~ 10 minutes. Then, 10 p M isoproterenol (ISO) with 10 p M phentolamine (PHE) was applied and steady state pH ; increased by ~ 0.21 pH units after a short delay of ~ 4 minutes. B. Two acid loads were performed during perfusion with Ca2+-free, HEPES-buffered medium. The initial acid load was imposed under control conditions and the second acid load and subsequent recovery of pH ; was conducted in the presence of 10 p M isoproterenol (ISO) with 10 p M phentolamine (PHE). The rates of pH; recovery from the intracellular acid load imposed under the influence of 10 pM isoproterenol (ISO) with 10 p M phentolamine (PHE) were increased by an average of 132% when compared to the control rates of recovery at all corresponding levels of pHj. Each trace represents data collected from different neurons. 126 0 [Ca z +] 0 + 200 uM EGTA 10 uM ISO + 10 uM PHE 3 6 9 12 15 18 21 24 27 Time (min) 0 [Caz ] 0 + 200 uM EGTA 10 UMIS0 + 10 (iM PHE N H 4 + N H 4 + 6 12 18 24 30 Time (min) 127 Figure 34. Effect of isoproterenol on steady state intracellular Ca 2 + during perfusion with Ca2+-free, HEPES-buffered medium A . The fluorescent indicator, fura-2, was utilized to measure changes in the BI334/BI3S0 (background corrected I334/I3m) value. During perfusion with Ca2+-free, HEPES-buffered medium, containing 200 uM EGTA, the average steady state BI33JBI3W value of ~ 0.40 decreased to ~ 0.31 which indicated a decrease in intracellular [Ca2+]. After [Ca 2 +]i attained a new resting level, 10 u M isoproterenol (ISO) with 10 u M phentolamine (PHE) was applied for ~ 12 minutes and had no effect on the resting BI32JBI3M levels. BI334/BI3S0 * 2+ values returned to the previous resting level upon re-introduction of external Ca . The trace represents the mean of data collected simultaneously from 9 cultured post-natal hippocampal neurons on a single coverslip. B. The adult hippocampal neuron was exposed to Ca2+-free, HEPES-buffered medium, containing 200 u M EGTA, and the mean resting value of BI334/BIJi0 fell to ~ 0.34, indicating a decrease in intracellular [Ca2 +]. Upon [Ca2+]j attaining a new steady level, 10 uM isoproterenol (ISO) and 10 u M phentolamine (PHE) were applied. BI334IBI3W values and, thus, resting intracellular [Ca2+] were unaffected by the P-adrenergic receptor agonist. 128 B. 0 [Ca2 +]0+ 200 uM EGTA 129 Figure 35. Effects of Sp-cAMPS on steady state pHj and on the rates of pH ; recovery from an imposed acid load during perfusion with HEPES-buffered medium A. From an initial resting level of pH; ~ 7.19, 25 p M Sp-cAMPS evoked a cytosolic alkalinization of ~ 0.24 pH units after a delay of ~ 6 minutes. B. The initial acid load was imposed under control conditions. Following recovery of pH i ; the neuron was pre-treated with 25 p M Sp-cAMPS for 3 minutes before the second acid load. The rates of recovery of pH, from the subsequent intracellular acidification, in the presence of 25 p M Sp-cAMPS, increased by an average of 221% when compared to the control rates of pH r recovery at the same absolute levels of pH;. Each trace represents data collected from a different neuron. 130 Time (min) 131 Figure 36. Effects of Rp-cAMPS on norepinephrine-induced actions on steady state pH; and on the rates of pH ; recovery from an imposed acid load during perfusion with HEPES-buffered medium A. A n adult hippocampal CA1 neuron was pre-treated with 50 p M Rp-cAMPS for ~ 18 minutes. A subsequent application of 10 p M norepinephrine (NE) failed to evoke an increase in resting pH; in the continued presence of 50 p M Rp-cAMPS. B. The initial acid load was performed under control conditions. Upon recovery of pHj the neuron was pre-treated for ~ 10 minutes before the second acid load with 50 p M Rp-cAMPS. The rates of pH ; recovery from an imposed intracellular acid load, under the influence of 10 p M norepinephrine (NE) and 50 p M Rp-cAMPS, were not significantly different to the control rates of pH; recovery when compared at all corresponding levels of pHj. Each trace represents data recorded from different neurons. 132 A . 7.6 7.5 ffi 7.4 a* 7.3 50 uM Rp-cAMPS 10 p M N E 7.2 10 15 20 Time (min) 25 30 B. 8.6 8.2 Sri 7.8 -7.4 N H , + 0 3 6 50 uM Rp-cAMPS l O p M N E N H , + 12 15 18 21 24 27 Time (min) 133 Figure 37. Effects of H-89 on norepinephrine-induced effects on steady state pH s and on the rates of pH ; recovery from an imposed acid load during perfusion with HEPES-buffered medium A . 10 p M H-89 was applied for ~ 9 minutes before the neuron was exposed to 10 u M norepinephrine. Under these conditions, 10 uM norepinephrine (NE) failed to evoke a rise in steady state pH,. B. An initial acid load was performed under control conditions. Following the recovery of pH i 5 10 uM of the protein kinase A inhibitor was applied 3 minutes before the start of the second acid load maneuver. The rates of pH ; recovery in the presence of 10 u M norepinephrine (NE) and 10 uM H-89 were not significantly different to the control rates of recovery after an imposed intracellular acid load at the same absolute levels of pH;. Each trace represents data collected from different neurons. 134 Time (min) 135 Discussion In peripheral cell types, external agents such as neurotransmitters can alter the activity of pHj regulating mechanisms. The possibility that such an interaction may occur in mammalian central neurons had previously not been investigated. This thesis describes the ability of norepinephrine to modulate the activity of the Na7H + exchanger, the principal acid extrusion mechanism operating in adult rat hippocampal CA1 neurons. The signal transduction system mediating the effects of norepinephrine on resting pH ; and on the rates of pH; recovery following an imposed acid load included P-adrenergic receptors, G s a , cAMP and protein kinase A (PKA). Indeed, in the absence of norepinephrine, both the activation of adenylyl cyclase (which leads to an accumulation of cytosolic cAMP) and the activation of P K A led to the subsequent modulation of the activity of the exchanger. Acid extrusion mechanisms present in adult rat hippocampal CA1 neurons In acutely dissociated adult rat hippocampal CA1 neurons, a Na+-dependent, HC03"-independent acid extrusion mechanism, most likely to be a Na7H + exchanger, was the principal acid extrusion mechanism operating during perfusion with HCOy-free HEPES-buffered medium. This variant of the Na7H + exchanger, as other laboratories have previously reported, is insensitive to a potent analogue of amiloride, EIPA (Raley-Susman et al., 1991; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al., 1996). During perfusion with HC037C02-buffered medium, Na7H + exchange still dominated acid extrusion, but a DIDS-sensitive, Na+-dependent HC037C1" exchanger also 136 contributed to acid extrusion and the maintenance of resting pHj. The activity of the anion exchanger was only evident in neurons with pH ( < 7.3. Schwiening and Boron (1994) also observed in acutely dissociated neonatal rat hippocampal CA1 neurons that the activity of a Na+-dependent HCOy/CT exchanger appeared to be regulated by the level of pHj. They concluded that the anion exchanger was the major acid extrusion mechanism in neonatal rat hippocampal CA1 neurons. However, the average resting pHj of their neurons was pHj 6.81 during perfusion with HC03"-free, HEPES-buffered medium and pH; 7.03 in the presence of HC037C02-buffered medium. The difference in steady state pH; levels observed by Schwiening and Boron (1994) and in the present study (mean resting pH; during perfusion with HC0 3/C0 2-buffered medium was pH ; 7.20) accounts for my placing a lesser emphasis on the role of the Na+-dependent HCOy/CT exchanger in the regulation of acid extrusion and steady state p H in rat hippocampal CA1 neurons. Furthermore, Schwiening and Boron (1994) did not investigate the possibility that, in neurons with higher resting levels of pH i 5 acid extrusion mechanisms other than the Na+-dependent HC0 37C1" exchanger may also participate in the maintenance of steady state pHj. M y results are, however, in complete agreement with those presented in a recent report by Bevensee et al. (1996) who performed a detailed study of the acid extrusion mechanisms involved in the regulation of pH { in acutely isolated mature (21- to 30 - day old) rat hippocampal CA1 neurons. Bevensee and colleagues (1996) found that, during perfusion with HEPES-buffered saline, hippocampal CA1 neurons had a broad range of resting pH ; values (6.3-7.7) and the distribution of the resting values of pH, was best-fitted with the sum of two Gaussian distributions with means at pH ; 6.68 and at pH; 137 7.32, respectively. Furthermore, during perfusion with HEPES-buffered and H C O y /CC»2-buffered saline, acid extrusion was dominated by an amiloride-insensitive Na7H + exchanger. In addition, the increase of resting pH f upon exposure to a HC03'-containing saline was dependent upon the initial steady state pH ; value in HEPES-buffered medium and was dependent on the activation of the Na+-dependent HC037C1" exchanger. The present results confirm all of these features of the activity of acid extrusion mechanisms in rat hippocampal neurons. Results presented here are also entirely consistent with a previous report characterizing the acid extrusion mechanisms present in cultured fetal rat hippocampal neurons (Baxter and Church, 1996). Effects of norepinephrine on pH{ Norepinephrine increased steady state pHj and the rate of pHj recovery from an imposed intracellular acid load during perfusion with HEPES-buffered or H C 0 3 7 C 0 2 -buffered medium. The mean rise of resting pH ; and the average percentage increase in the rates of pH; recovery from an imposed acid load in the presence of norepinephrine during perfusion with either type of media were not statistically different. Therefore, the results suggest that norepinephrine is not modulating the activity of any HC03"-dependent acid extrusion mechanism(s) and is affecting exclusively the activity of the Na7H + exchanger. During perfusion with HEPES-buffered medium, the norepinephrine-induced effect on resting pH> was not mediated by an increase in [Ca2+]j but did require external Na + and appeared to be long-lasting as the increased level of pH ( was sustained for as long as stable recordings could be maintained (~ 30 min). The concentration of norepinephrine 138 (10 pM) utilized in the present study was consistent with the concentrations employed in a large number of studies of the effects of norepinephrine on hippocampal neuronal function (e.g. Stanton and Sarvey, 1985; Madison andNicoll, 1986a; Bergles et al, 1996; also see Harley et al, 1996). The effects of norepinephrine on steady state pH, and on the rate of pH ; recovery from an imposed intracellular acidification were mediated by both (3, and p 2 adrenergic receptors. The possible involvement of the p3-adrenoceptor subtype was not investigated as the localization of this adrenergic receptor protein in the hippocampus has not yet been fully determined (Giacobino, 1995; Summers et al, 1995). P-adrenoceptor-mediated increases in the activity of NaTET exchangers have been previously reported in fibroblast PS120 cells expressing the pNHE isoform (Borgese et al, 1994) and in canine enteric cells (Barber et al, 1989). In contrast, in sheep cardiac Purkinje fibres, p- (but not a-) adrenergic receptor activation inhibits NaTET exchange and, consequently, slows the rate of pH: recovery from an intracellular acidification and reduces resting pH; (Guo et al, 1992). And in rat ventricular myocytes (Iwakura et al, 1990; Terzic et al, 1992; Wallert and Frohlich, 1992) and in rat vascular smooth muscle cells (Owen, 1986) stimulation of Na7H + exchange, and the subsequent alkalinization of resting pHj, is mediated by oc r adrenergic receptors. It is possible that the effects of norepinephrine on steady state pH; and on the rate of pHj recovery from an imposed acid load may reflect, at least in part, a change in the buffering capacity of rat hippocampal neurons and may not be entirely due to an enhancement of the activity of the NaVFT exchanger. Thus, in the absence of any 139 changes in the activities of acid extrusion mechanisms, i f intracellular buffering power is reduced then dpH/d/ will be increased (Boron, 1989). However, in sheep cardiac Purkinje fibres, norepinephrine had no significant effect on the intrinsic buffering power as the catecholamine altered the extrusion rate of the Na7H + exchanger via p-adrenergic receptors and cAMP (Guo et al, 1992). In addition, Bevensee and his colleagues (1996) observed in single hippocampal CA1 neurons freshly dissociated from 21 - 30 day old rats that the rates of pH ; recovery from an imposed acid load of "high p H " (resting pHj > 7.05) neurons were higher compared to those of "low p H " (resting pH ; < 7.05) neurons. They concluded that these observations were not due to differences in buffering capacity between the two "types" of hippocampal CA1 neurons but reflected differences in acid-base fluxes mediated by the amiloride-insensitive Na7H + exchanger. They also suggested that the differences they observed in steady state pH; and net acid extrusion rates might very well be due to different functional states of the Na7H + exchanger or different levels of expression of the exchanger. In the present study, norepinephrine increased resting pH ; after only a short delay (~ 3 - 4 minutes) suggesting that increased expression of the exchanger is unlikely to be able to account for the increase in acid extrusion rates observed. Rather, the data appear consistent with a p-adrenergic receptor-mediated modulation of the activity of existing Na7H + exchangers. In the present study, the distribution of resting pH; of naive neurons, i.e. those which had not been exposed to norepinephrine or other P-adrenergic receptor agonists, was best fitted with the sum of two Gaussian distributions with means at pH ; 6.93 and pH, 7.44. Upon exposure to norepinephrine, isoproterenol, dobutamine or terbutaline, the 140 bimodal distribution of initial steady state pH; values of naive neurons was converted to a single Gaussian distribution centered at pHj 7.54 (see Fig. 21). In concert with the increase in resting pH i 5 norepinephrine and B-adrenergic agonists also increased the rate of acid extrusion via a long-term activation of the Na7H + exchanger. Thus, the results of the present study complement those reported by Bevensee et al. (1996) and, furthermore, suggest the possibility that P-adrenoceptor-mediated activation of the Na7H + exchanger may represent an additional mechanism whereby norepinephrine can regulate the functional capabilities of hippocampal CA1 neurons. Effects of norepinephrine on mammalian central neuronal excitability In 1970, Kety proposed that the functional role of norepinephrine in the central nervous system was as a mediator of the aroused state, in spite of the electrophysiological studies that indicated that norepinephrine appeared to inhibit cell firing (see Harley, 1987). Norepinephrine in Kety's model provided the substrate for memory of and attention to selective events. He argued that the enhancing effects of norepinephrine on a small number of neurons may not be detected by random recordings of unit activity (see Harley, 1987). Initial electrophysiological studies in the rat hippocampal slice preparation determined that an iontophoretic application of norepinephrine evoked a small hyperpolarization associated with a modest decrease in input resistance (Segal and Bloom, 1974). The effects were most pronounced and long-lasting when norepinephrine was applied directly on the pyramidal cell layer with the recording electrode in the soma. 141 Later studies involving the stimulation of the locus coeruleus-hippocampal pathway indicated that norepinephrine, acting via B-adrenergic receptors, enhanced both inhibitory and excitatory responses, effects which were attributed to an "improvement" of the signal to noise ratio in the hippocampus (Segal, 1981a). Further, it was determined that the actions of norepinephrine on hippocampal neurons depended on the types of adrenergic receptor-subtype that were involved (Mueller et al, 1982). Mueller et al. (1982) utilized pressure ejection of norepinephrine into the in vivo hippocampus and determined that norepinephrine could both enhance and reduce the evoked firing rate of hippocampal neurons via p - and a-adrenergic receptors, respectively. These early studies laid the foundation for the current, widely-held view that norepinephrine acts as a putative neuromodulator in the mammalian central nervous system, for example enhancing neuronal responsiveness to neurotransmitters such as excitatory amino acids (e.g. Mori-Okamato and Tatsuno, 1988; Dodt et al., 1991). Dolphin (1982) observed that norepinephrine, acting via both a and P adrenergic receptors, could modulate the K+-induced release of glutamate onto cerebellar Purkinje cells as well as potentiating the postsynaptic response of Purkinje cells to glutamate. Norepinephrine has also been determined to be an excitatory neuromodulator for neurons in layer V of the cat and rodent sensorimotor cortex due to its ability to reduce the slow afterhyperpolarization and subsequent enhancement of evoked field potentials (Foehring et al, 1989; Nowicky et al, 1992). In the rat dentate gyrus, norepinephrine and isoproterenol caused a long-lasting potentiation of evoked field potentials following stimulation of the medial perforant pathway which was then termed "norepinephrine-142 induced long-lasting potentiation" (Dahl and Sarvey, 1989; Stanton et al, 1989; Dahl and L i , 1994). In the rat amygdala, an application of isoproterenol caused a reduction of the after-hyperpolarizations (AHPs) mediated by Ca2+-activated K + conductances which led to an increased firing of action potentials (Huang et al, 1996). The effect was abolished with an intracellular application of a selective inhibitor of P K A (Huang et al, 1996). Another effect induced by isoproterenol in the rat amygdala was a sustained enhancement of the postsynaptic response to glutamate, similar to that observed in norepinephrine-induced long-lasting potentiation in the dentate gyrus (Gean et al, 1992). In the CA1 region of the hippocampus norepinephrine can influence both the excitability of neurons and also their response to various neurotransmitters. Initially, Mueller et al (1982) observed that P-adrenergic agonists and activation of the locus coeruleus-hippocampus pathway could increase the amplitude of evoked population spikes in the CA1 region. Norepinephrine, in rat pyramidal CA1 neurons, was shown to reduce AHPs via activation of P-adrenergic receptors and the increased production of intracellular cAMP (Haas and Konnerth, 1983; Madison and Nicoll, 1986a,b). The effect of norepinephrine on the AHPs was not dependent upon any changes in Ca 2 + influx (Haas and Konnerth, 1983; Madison and Nicoll, 1986a). The reduction of AHPs led to an increased frequency in the firing rate of CA1 pyramidal neurons and, thus, an increased level of excitability (Madison and Nicoll, 1986a). Heginbotham and Dunwiddie (1991) later described the ability of isoproterenol to increase the amplitude of the evoked population spike (but not of the field excitatory postsynaptic potential (fEPSP)) in the CA1 region of the rat hippocampal slice preparation. The long-lasting effect of the P-143 adrenergic receptor agonist was not terminated by prolonged washout of isoproterenol or by subsequent application of a P-adrenoceptor antagonist. This postsynaptic effect was determined to be mediated by an activation of adenylyl cyclase and the subsequent increase in cytosolic [cAMP] (Heginbotham and Dunwiddie, 1991; Dunwiddie et al, 1992). As well as affecting hippocampal neuronal function via post-synaptic mechanisms, recent reports have suggested that P-adrenergic receptor activation may also act presynaptically to enhance synaptic transmission (e.g. Gereau and Conn, 1994a). As previously stated, norepinephrine can also influence some of the responses of hippocampal neurons to other neurotransmitters. Activation of p-adrenergic receptors, for example, can markedly potentiate the effects of metabotropic glutamate receptor agonists on hippocampal CA1 pyramidal neuron excitability (Gereau and Conn, 1994b). This effect of norepinephrine can be long-lasting and persists even after cAMP levels have returned to baseline (Gereau et al., 1995). To summarize, norepinephrine may regulate neuronal excitability in opposing directions depending on the adrenergic receptor involved, and may also modulate the actions of other external agents on the excitability of various types of neurons. Signal transduction systems andpHt Components of the second messenger system that are classically been linked to p-adrenergic receptors, specifically cAMP and protein kinase A, were also implicated in the norepinephrine-induced change in pH; detailed in this study. There are several reports of consensus sequences for protein phosphorylation present on the cytoplasmic tail of all 144 known isoforms of the Na7H + exchanger (Bianchini et al, 1991; Sardet et al, 1991; Guizouarn et al, 1993; Winkel et al, 1993; Bertrand et al, 1994). This implies that the activity of all Na7H + exchangers, including the Na7H + exchanger found in rat hippocampal neurons, may be dependent on the state of phosphorylation of the exchanger. Indeed, in the present study, both forskolin, an activator of adenylyl cyclase, and Sp-cAMPS, an activator of P K A , increased resting pH ; and the rate of pHj recovery from an imposed intracellular acidification. In addition, when the hippocampal neurons were pre-treated with inhibitors of adenylyl cyclase or P K A , the norepinephrine-induced effects on steady state pHj and on the rate of pH, recovery from an imposed acid load were occluded. However, the results of the present study did not directly identify P K A as the final step in the pathway mediating the actions of norepinephrine on the activity of the Na7H + exchanger, i.e. phosphorylation of the NaYFT exchanger by P K A . Experiments involving protein phosphatase inhibitors, e.g. okadaic acid, were not performed because protein phosphatases are not necessarily selective for the actions of specific protein kinases such as P K A (Cohen and Cohen, 1989; Hunter, 1995; Wera and Hemmings, 1995). Therefore, the effect of a given protein phosphatase inhibitor (if any) on the activity of the Na7H + exchanger in rat hippocampal neurons would not necessarily provide additional evidence for the involvement of PKA-mediated phosphorylation in the regulation of the exchanger. Nevertheless, characterization of the effects of protein phosphatases inhibitors on the activity of the rat hippocampal neuron Na7H + exchanger would represent an interesting area of future study. The activity of the Na7H + exchanger in rat hippocampal neurons could be modulated by several signal transduction pathways, 145 i.e. the exchanger could be phosphorylated by many types of protein kinases to modulate its acid extrusion rate, as is known to be the case for Na7H + exchangers in other cell types (see Frelin et al, 1988). Therefore, the present study indicates solely that the activation of G s a , adenylyl cyclase, and P K A are the minimum requirements for mediating the norepinephrine-induced, P-adrenoceptor-mediated alteration of the activity of the NaTFT exchanger in rat hippocampal CA1 neurons. Similarly, the adenylyl cyclase/PKA system has been identified as participating in the modulation of the activity of NaTFT exchangers in a variety of cell types (e.g. Clark et al, 1990; Green and Kleeman, 1992; Borensztein et al, 1993; Guizouarn et al, 1993; Azarani et al, 1995; Kandasamy et al, 1995). For example, in cardiac Purkinje fibres and in various epithelial systems, a rise in cytosolic [cAMP] reduces Na7H + exchange activity (Reuss and Petersen, 1985; Semrad and Chang, 1987; Guo et al, 1992; Wu and Vaughan-Jones, 1994). In rabbit kidney brush-border membranes, oppossum kidney cells and rat medullary thick ascending limb cells, cAMP-mediated inhibition of the activity of Na7H + exchangers involves the activation of P K A (Weiman et al, 1987; Kahn et al, 1985; Clark et al, 1990; Borensztein et al, 1993). In contrast, Borgese et al. (1994) reported that the trout erythrocyte PNHE isoform of the NaTFT exchanger could be activated by both norepinephrine and cAMP (see also Guizouran et al, 1993). The pathway mediating the activation of the PNHE isoform of the NaTFT exchanger is therefore apparently similar to the pathway described in the present study. Borgese et al. (1994) also observed a delay in the activation of the NaTFT exchanger and maximum activity was achieved 9-12 minutes after initial treatment with norepinephrine. Gupta et 146 al. (1992) noted that an application of forskolin caused an increase in steady state pHj in UMR-106 cells via modulation of the activity of the NaTFT exchanger. This effect on the extrusion rate of the cation exchanger in the osteoblastic cell line was blocked by H-89 thereby implicating the activation of P K A in the forskolin-induced increase of resting pHi. Kandasamy et al. (1995) reported that rat NaTH + exchanger isoforms (NHE-1, -2 and -3) that were stably expressed in AP-1 cells devoid of endogenous NaTH + exchange activity are differentially responsive to cAMP-raising agents. Upon increasing cytosolic [cAMP] and the subsequent activation of P K A , the activities of NHE-1 and -2 were enhanced while the extrusion rate of the NHE-3 isoform was reduced (Kandasamy et al., 1995). The present results do not exclude the possibility that the activity of the NaTH + exchanger in rat hippocampal CA1 neurons may also be regulated by additional effector systems, such as the phospholipase C/protein kinase C system or the Ca2+-activated, calmodulin-dependent protein kinase II system (see Introduction). For example, the P-adrenoceptor-mediated regulation of the activity of the NaTH + exchanger in enteric canine and astrocytoma cells is independent of changes in cAMP accumulation (Barber et al, 1989). In summary, norepinephrine can alter the activity of the NaTH + exchanger in rat hippocampal neurons via a specific second messenger pathway. In addition, the components of this intracellular pathway, specifically cAMP and protein kinase A , can themselves affect the extrusion rate of the exchanger. 147 Effects of activation of adenylyl cyclase and protein kinase A on neuronal excitability Chavez-Noreiga and Stevens (1992) reported that in the CA1 region of rat hippocampal slices, forskolin potentiates the fEPSP evoked by the stimulation of the Schaffer collateral/commissural pathway. They interpreted these results as being due to an enhancement of synaptic efficacy or strength at Schaffer collateral-CAl pyramidal neuron synapses in the rat hippocampus. Later, it was determined that presynaptic mechanisms such as an enhanced spontaneous and evoked release of neurotransmitter were the main contributors to the increase in synaptic efficacy produced by forskolin (Chavez-Noriega and Stevens, 1994). Forskolin also enhances the synaptic strength of mossy fiber synapses in the CA3 region of the rat hippocampal slice (Hopkins and Johnston, 1988). In the dentate gyrus of the rat hippocampus, forskolin enhanced the norepinephrine-induced potentiation of the granule cell population spike evoked by perforant path stimulation (Stanton and Sarvey, 1985). Using whole-cell recordings, activation of P K A was shown to enhance the response of cultured hippocampal pyramidal neurons to glutamate via modulation of the activity of non-NMDA receptors (Greengard et al, 1991). Therefore, both cAMP and protein kinase A have been shown to enhance the synaptic response to afferent stimulation and also to modulate the actions of other transmitters such as glutamate on hippocampal neurons. 148 Physiological significance of norepinephrine- and second messenger-induced changes in pHi The functional significance of both the norepinephrine- and second messenger-induced modulation of the activity of the Na + /H + exchanger in rat hippocampal neurons is unknown. Nevertheless, there are a number of possible implications of the results reported in this thesis for central neuronal function. Firstly, some of the reported actions of norepinephrine in hippocampal neurons may, at least in part, be mediated by an alkalinization of resting pHj. Secondly, other external agents that also evoke a rise in c A M P and/or activate protein kinase A, may also alter resting pHj to, in turn, mediate at least some of their respective actions in neurons. And finally, norepinephrine-induced effects on the activity of the Na + /H + exchanger may allow hippocampal neurons to alleviate more effectively acid loads evoked by other transmitters. As described above, many of the P-adrenoceptor-mediated actions of norepinephrine in the hippocampal formation, notably in the CA1 subfield, involve modulation of the excitability of these neurons and also of their responses to other neurotransmitters such as glutamate. Therefore, the novel finding that norepinephrine, through the activation of P-adrenergic receptors, evokes a long-lasting increase in steady state pHj via an enhancement of the proton extrusion rate of the Na7H + exchanger may offer a possible mechanism for some of the reported p-adrenoceptor-mediated actions of norepinephrine in the rat hippocampus. For example, it is well documented that in various types of neurons, including hippocampal CA1 cells, an intracellular alkalosis can affect a variety of ionic conductances and thereby lead to an increase in cell excitability 149 (e.g. Moody, 1984; Church, 1992; Daumas and Andersen, 1993; Mironov, 1995; Cowan and Martin, 1996). Thus, it is possible that the norepinephrine-induced increase in steady state pH ; may be mediating some of the effects of norepinephrine on the excitability of hippocampal CA1 neurons. For example, studies by Dunwiddie and colleagues (1991 and 1992) indicated that norepinephrine, acting via B-adrenergic receptors, caused a long-lasting potentiation of the evoked population spike in the CA1 region that was dependent on an increase in cytosolic [cAMP]. They reported that the potentiation of the evoked population spike outlasted the duration of the application of isoproterenol, i.e. the mean population spike response was still significantly enhanced following 30 minutes of washout of isoproterenol. In the present study, norepinephrine caused a long-lasting increase in resting pH ; that was also cAMP-dependent, suggesting the possibility that a norepinephrine-induced increase in steady state pHj may participate in the effects of norepinephrine on the evoked population spike in hippocampal CA1 pyramidal neurons. The effects of norepinephrine, cAMP, and protein kinase A on steady state pH; may also affect neuronal excitability by modulating gap junctional conductances, which are known to be sensitive to changes in pH; (Spray and Bennett, 1985; Mac Vicar and Jahnsen, 1985; Church and Baimbridge, 1991; Jefferys, 1995). Interestingly, B-adrenoceptor agonists increase gap junctional intercellular communication between gastric epithelial cells; the effect is mediated by an increase in cAMP and a subsequent activation of the Na7H + exchanger present in these cells (Ueda et al., 1994; see also Flagg-Newton et al., 1981). It would be interesting to determine whether B-adrenergic 150 receptor agonists affect electrotonic coupling (or Lucifer Yellow dye coupling) between rat hippocampal CA1 pyramidal neurons. In contrast, Madison and Nicoll (1986a) noted that norepinephrine, acting via P-adrenergic receptors, reduced the AHPs mediated by Ca2+-activated K + conductances in CA1 pyramidal neurons. However Church (1992) observed in CA1 pyramidal neurons in an hippocampal slice preparation that reductions in pHj at a constant pH 0 also reduced AHPs mediated by Ca2+-activated K + conductances, whereas raising pH ; enhanced AHPs (also see Church and McLennan, 1989). An increase in resting pH, evoked by norepinephrine acting via P-adrenoceptors might therefore be expected to enhance AHPs whereas, in fact, the opposite is observed. This apparent discrepancy may, however, be reconciled by the possibility that proton channels could be located within "clusters" of Ca 2 + and K + channels and may create a pHrregulating micro-environment that would selectively modulate the activity of the AHPs (see Meech and Mackie, 1993). This example serves to highlight the fact that the effects of changes in pH; on neuronal function are not necessarily straightforward. Other neurotransmitters in the hippocampus that are positively linked to cAMP production are able to modulate the excitability of hippocampal neurons. In the CA1 area of the rat hippocampus, serotonin acting at postsynaptic 5-HT4 receptors can reduce AHPs and thereby increase neuronal excitability (Siarey et al, 1995). Histamine can modulate the synaptic responses of hippocampal CA3 and CA1 pyramidal neurons to afferent stimulation, increase the magnitude of the evoked population spike and enhance NMDA-mediated synaptic transmission (Segal, 1981b; Bekkers, 1993). Many of the 151 excitatory actions of histamine are mediated by its H 2 receptor subtype which is positively linked to the adenylyl cyclase/cAMP/PKA signal transduction system (Yanovsky et al, 1995). And, as with norepinephrine, the effects of histamine on hippocampal CA1 and CA3 neurons have been shown to outlast the presence of histamine in the tissue (Haas and Green, 1986). Future studies should address the possibility that other neuromodulators, such as histamine, may be able to regulate the activity of pH; regulating mechanisms in mammalian central neurons. Another possibility arising from the results of the present study is that norepinephrine, by increasing the rate of acid extrusion, may increase the ability of hippocampal CA1 neurons to dissipate intracellular acid loads imposed by pathophysiological events or by other neurotransmitters such as glutamate. For example, in cultured fetal and post-natal hippocampal neurons, glutamate evokes a fall in resting pHi of 0.30 - 0.50 pH units (Hartley and Dubinsky, 1993; Irwin et al, 1994; Wang et al, 1994). The marked decrease in resting pH; could have many consequences, such as alteration of the activity of critical metabolic enzymes (e.g. brain phosphofructokinase), and may act synergistically with increases in [Ca2+]i to contribute to excitotoxic and ischemic neuronal death (Tombaugh and Sapolsky, 1990; Koch and Barish, 1991; Nedergaard et al, 1991; Wang et al, 1994). The ability of norepinephrine to increase the acid extrusion rate of the Na7H + exchanger, the principal acid extrusion mechanism operating in adult rat hippocampal CA1 neurons, may represent an important mechanism whereby neurons are able to alleviate rapidly the intracellular acidification evoked by 152 glutamate and may thereby reduce the possibility of cell damage due to prolonged exposure to an acidified cytoplasm. Finally, although the present study focused on the effects of norepinephrine on pHi, the resultant cytosolic alkalinization may also affect extracellular pH. During perfusion with HEPES-buffered medium, norepinephrine increased resting pHj by an average of 0.27 pH units which would indicate that protons were being actively extruded from hippocampal neurons. The resultant local (surface) acidification of the extracellular space could affect nearby glutamate receptors, specifically the N M D A receptor subtype (Tang et al, 1990; Traynelis and Cull-Candy, 1990), as well as voltage-gated conductances (e.g. Tombaugh and Somjen, 1996). Indeed, it has been established that the extracellular alkaline transients which are observed consequent upon N M D A receptor-mediated falls in pHj are able to modulate N M D A receptor function in a physiologically relevant time frame (e.g. Gottfried and Chesler, 1994; see Introduction). Therefore, not only could the rise in resting pHj due to norepinephrine affect cell excitability directly but the resultant extracellular acidosis may also influence the excitability of hippocampal neurons. This is the first known report of an external agent modulating the activity of a pH; regulating mechanism in the mammalian central nervous system. As a neuromodulator, norepinephrine acts to modulate excitatory and inhibitory inputs to neurons and thereby has significant influence on the functional state of target regions such as the hippocampus. The ability of norepinephrine, cAMP and P K A to induce a long-lasting 153 increase in pHj may have significant effects on the excitability of neurons and may represent a possible mechanism for a number of the reported actions of norepinephrine and of c A M P and P K A in mammalian central neurons. Given the complex actions of both norepinephrine and pH, on the excitability of hippocampal neurons, the ability of norepinephrine to regulate the activity of the Na7H + exchanger may represent a powerful pathway that could not only influence directly the activity of these neurons but also modulate the effects of other neurotransmitters on hippocampal CA1 neurons. 154 References Amos, B. J. and Richards, C. D. (1994). Effect of glutamate on intracellular pH of rat neurones maintained in culture. Journal of Physiology 479.P, 43P. Andreeva, N., Khodorov, B., Stelmashook, E., Sokolova, S., Cragoe, E. and Victorov, I. (1992). 5-(7Y-ethyl-A'-isopropyl)amiloride and mild acidosis protect cerebellar granule cells against glutamate-induced delayed neuronal death. Neuroscience 49, 175-181. Aram, J. A. and Lodge, D. (1987). Epileptiform activity induced by alkalosis in rat neocortical slices: Block by antagonists of 7Y-methyl-D-asparate. Neuroscience Letters 83, 345-350. Aronson, P. S. (1985). Kinetic properties of the plasma membrane Na + -H + exchanger. Annual Review of Physiology 47, 545-560. Azarani, A., Goltzman, D. and Orlowski, J. (1995). Parathyroid hormone and parathyroid -related peptide inhibit the apical Na7H+ exchanger NHE-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C. Journal of Biological Chemistry 270, 20004-20010. Baker, P. F. and Honerjager, P. (1978). Influence of carbon dioxide on level of ionised calcium in squid axons. Nature 273, 160-161. Balestrino, M. and Somjen, G. G. (1988). Concentration of carbon dioxide, interstitial pH and synaptic transmission in hippocampal formation of the rat. Journal of Physiology 396, 247-266. Barber, D. L. and Ganz, M. B. (1992). Guanine nucleotides regulate B-adrenergic activation of Na-H exchange independently of receptor coupling to G s. Journal of Biological Chemistry 267, 20607-20612. Barber, D. L., Ganz, M. B., Bongiorno, P. B. and Strader, C. D. (1992). Mutant constructs of the B-adrenergic receptor that are uncoupled from adenylyl cyclase retain functional activation of Na-H exchange. Molecular Pharmacology 41, 1056-1060. Barber, D. L., McGuire, M. E. and Ganz, M. B. (1989). P-adrenergic and somatostatin receptors regulate Na-H exchange independent of cAMP. Journal of Biological Chemistry 264, 21038-21042. Baxter, K. A. (1995). M. Sc. thesis. Department of Anatomy, University of British Columbia. 155 Baxter, K. A . and Church, J. (1996). Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. Journal of Physiology 493.2, 457-470. Bazaes, S. E. and Kemp, R. G. (1990). Resistance of brain phosphofructo-1-kinase to pH-dependent inhibition. Metabolic Brain Disease 5, 111-118. Bekkers, J. M . (1993). Enhancement by histamine of NMDA-mediated synaptic transmission in the hippocampus, Science 261, 104-106. Bergles, D. E., Doze, V . A. , Madison, D. V . and Smith, S. J. (1996). Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons. Journal of Neuroscience 16, 572-585. Bertrand, B., Wakabayashi, S., Ikeda, T., Pouyssegur, J. and Shigekawa, M . (1994). The Na7H + exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Journal of Biological Chemistry 269, 13703-13709. Bevensee, M . O., Cummins, G. G., Boron, W. F. and Boyarsky, G. (1996). pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. Journal of Physiology 494.2, 315-328. Bevensee, M . O., Schwiening, C. J. and Boron, W. F. (1995). Use of B C E C F and propidium iodide to assess membrane integrity of acutely isolated CA1 neurons from the rat hippocampus. Journal of Neuroscience Methods 58, 61-75. Bianchini, L. , Woodside, M . , Sardet, C , Pouyssegur, J., Takai, A . and Grinstein, S. (1991). Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na7H + antiport. Journal of Biological Chemistry 266, 15406-15413. Booze, R. M . , Crisostomo, E. A. and Davis, J. N . (1989). Species differences in the localization and number of CNS beta adrenergic receptors: Rat versus guinea pig. Journal of Pharmacology and Experimental Therapeutics 249, 911-920. Borensztein, P., Juvin, P., Vernimmen, C , Poggioli, J., Paillard, M . and Bichara, M . (1993). cAMP-dependent control of Na7H + antiport by A V P , PTH and PGE 2 in rat medullary thick ascending limb cells. American Journal of Physiology 264, F354-F364. Borgese, F., Malapert, M . , Fievet, B., Pouyssegur, J. and Motais, R. (1994). The cytoplasmic domain of the Na7H + exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHEl/trout (VNHE antiporter. Proceedings of the National Academy of Science, U.S.A. 91, 5431-5435. 156 Borrnann, J., Hamill, O. P. and Sakmann, B. (1987). Mechanism of amino permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. Journal of Physiology 385, 243-248. Boron, W. F. (1989). Cellular buffering and intracellular pH. In: The Regulation of Acid-Base Balance, pp. 33-56. Eds. D. W. Seldin and G. Giebisch. Raven Press. Boron, W. F. and De Weer, P. (1976). Intracellular pH transients in squid giant axons caused by CO2, N H 3 and metabolic inhibitors. Journal of General Physiology 67, 91-112. Boyarsky, G., Ganz, M . B., Sterzel, R. B. and Boron, W. F. (1988). pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3". American Journal of Physiology 255, C844-C856. Bright, G. R., Fisher, G. W., Rogowska, J. and Taylor, D. L . (1987). Fluorescence ratio imaging microscopy: Temporal and spatial measurements of cytoplasmic pH. Journal of Cell Biology 104, 1019-1033. Bright, G. R., Fisher, G. W., Rogowska, J. and Taylor, D. L . (1989). Fluorescence ratio imaging microscopy. Methods in Cell Biology 30, 157-192. Busa, W. B. (1986). Mechanisms and consequences of pH-mediated cell regulation. Annual Review of Physiology 48, 389-402. Busa, W. B. and Nuccitelli, R. (1984). Metabolic regulation via intracellular pH. American Journal of Physiology 246, R409-R438. Bylund, D. B., Eikenberg, D. C , Hieble, J. P., Langer, S. Z., Lefkowitz, R. J., Minneman, K . P., Molinoff, P. B., Ruffolo, R. R. and Trendelenburg, U . (1994). IV. International union of pharmacology nomenclature of adrenoceptors. Pharmacological Reviews 46, 121-136. Cano, A. , Miller, R. T., Alpern, R. J. and Preisig, P. A . (1994). Angiotensin II stimulation of Na-H antiporter activity is cAMP independent in OKP cells. American Journal of Physiology 266, C1603-C1608. Cantacuzene, D., Kirk, K. L. , McCulloh, D. H . and Creveling, C. R. (1979). Effect of fluorine substitution on the agonist specificity of norepinephrine. Science 204, 1217-1219. Carafoli, E. (1987). Intracellular Ca homeostasis. Annual Review of Biochemistry 56, 395-433. Carbone, E., Testa, P. L. and Wanke, E. (1981). Intracellular pH and ionic channels in the Loligo vulgaris giant axon. Biophysical Journal 35, 393-413. 157 Cash, R., Raisman, R., Lanfumey, L., Ploska, A . and Agid, Y . (1986). Cellular localization of adrenergic receptors in human and rat brain. Brain Research 370, 127-135. Cassel, D. and Pfeuffer, T. (1978). Mechanism of cholera toxin: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proceedings of the National Academy of Science, U.S.A. 75,2669-2673. Chaillet, J. R. and Boron, W. F. (1985). Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. Journal of General Physiology 86, 765-794. Chavez-Noriega, L. E. and Stevens, C. F. (1992). Modulation of synaptic efficacy in field CA1 of the rat hippocampus by forskolin. Brain Research 574, 85-92. Chavez-Noriega, L. E. and Stevens, C. F. (1994). Increased transmitter release at excitatory synapses produced by direct activation of adenylate cyclase in rat hippocampal slices. Journal of Neuroscience 14, 310-317. Chen, J. C. T. and Chesler, M . (1992a). Modulation of extracellular pH by glutamate and G A B A in rat hippocampal slices. Journal of Neurophysiology 67, 29-36. Chen, J. C. T. and Chesler, M . (1992b). Extracellular alkaline shifts in rat hippocampal slice are mediated by N M D A and non-NMDA receptors. Journal of Neurophysiology 68, 342-344. Chesler, M . (1986). Regulation of intracellular pH in reticulospinal neurones of the lamprey, Petromyzon marinus. Journal of Physiology 381, 241-261. Chesler, M . (1990). The regulation and modulation of pH in the nervous system. Progress in Neurobiology 34, 401 -427. Chesler, M . and Kaila, K. (1992). Modulation of pH by neuronal activity. Trends in Neuroscience 15, 396-402. Chijiwa, T., Mishima, A. , Hagiwara, M . , Sano, M . , Hayashi, K. , Inoue, T., Naito, K. , Toshioka, T. and Hayashi, H. (1990). Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, A^-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PCI 2D pheochromocytoma cells. Journal of Biological Chemistry 265, 5267-5272. Choi, D. W. (1998). Calcium-mediated neurotoxicity: Relationship to specific channel type and role in ischemic damage. Trends in Neuroscience 11, 465-469. 158 Church, J. (1992). A change from H C 0 3 - - C 0 2 - to HEPES-buffered medium modifies membrane properties of rat CA1 pyramidal neurones in vitro. Journal of Physiology 455,51-71. Church, J. and Baimbridge, K. G. (1991). Exposure to high-pH medium increases the incidence and extent of dye coupling between rat hippocampal CA1 pyramidal neurons in vitro. Journal of Neuroscience 11, 3289-3295. Church, J. and McLennan, H. (1989). Electrophysiological properties of rat CA1 pyramidal neurones in vitro modified by changes in extracellular bicarbonate. Journal of Physiology 415, 85-108. Clark, J. D. and Limbird, L. E. (1991). N a + - H + exchanger subtypes: A predictive review. American Journal of Physiology 261, C945-C953. Clark, J. D., Cragoe, E. J. and Limbird, L. E. (1990). oc2-adrenergic receptors regulate Na + -H + exchange via a cAMP-dependent mechanism. American Journal of Physiology 259, F977-F985. Cohen, P. and Cohen, P. T. W. (1989). Protein phosphatases come of age. Journal of Biological Chemistry 264, 21435-21438. Cowan, A . I. and Martin, R. L. (1996). Ionic basis of the membrane potential responses of rat dorsal vagal motoneurones to HEPES buffer. Brain Research 111, 69-75. Dahl, D. and L i , J. (1994). Induction of long-lasting potentiation by sequenced applications of isoproterenol. NeuroReport 5, 657-660. Dahl, D. and Sarvey, J. M . (1989). Norepinephrine induces pathway-specific long-lasting pontentiation and depression in the rat hippocampal dentate gyrus. Proceedings of the National Academy of Science, U.S.A. 86,4776-4780. Daly, J. W., Padgett, W. and Seamon, K. B. (1982). Activation of cyclic A M P -generating systems in brain membranes and slices by the diterpene forskolin: Augmentation of receptor-mediated responses. Journal of Neurophysiology 38, 532-544. Daumas, P. and Andersen, O. S. (1993). Proton block of rat sodium channels. Journal of General Physiology 101, 27-43. Deitmer, J. and Schlue, W.-R. (1987). The regulation of intracellular pH by identified glial cells and neurons in the central nervous system of the leech. Journal of Physiology 388, 261-283. Desilets, M . , Puceat, M . and Vassort, G. (1994). Chloride dependence of pH modulation by B-adrenergic agonist in rat cardiomyocytes. Circulation Research 15, 862-869. 159 Dhanasekaran, N . , Prasad, M . V . V . S. V. , Wadsworth, S. J., Dermott, J. M . and van Rossum, G. (1994). Protein kinase C-dependent and -independent activation of Na + /H + exchanger by Goc12 class of G proteins. Journal of Biological Chemistry 269, 11802-11806. Dixon, D. B. , Takahashi, K.-I. and Copenhagen, D. R. (1993). L-glutamate suppresses H V A calcium current in catfish horizontal cells by raising intracellular proton concentration. Neuron 11, 267-277. . Dodt, H.-U., Pawelzik, H. and Zieglgansberger, W. (1991). Actions of noradrenaline on neocortical neurons in vitro. Brain Research 545, 307-311. Doering, A. E. and Lederer, W. J. (1993). The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. Journal of Physiology 466, 481-499. Dolphin, A . C. (1982). Noradrenergic modulation of glutamate release in the cerebellum. Brain Research 252, 111-116. Dolphin, A . C , Hamon, M . and Blockaert, J. (1979). The resolution of dopamine and p, and p2-adrenergic sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex. Brain Research 179, 305-317. Duncan, G. E., Little, K. Y. , Koplas, P. A., Kirkman, J. A. , Breese, G. R. and Stumpf, W. E. (1991). P-adrenergic receptor distribution in human and rat hippocampal formation: Marked species differences. Brain Research 561, 84-92. Dunwiddie, T. V . , Taylor, M . , Heginbotham, L. R. and Proctor, W. R. (1992). Long-term increases in excitability in the CA1 region of rat hippocampus induced by P-adrenergic stimulation: Possible mediation by cAMP. Journal of Neuroscience 12, 506-517. Ebine, Y . , Fujiwara, N . and Shimoji, K. (1994). Mild acidosis inhibits the rise in intracellular C a 2 + concentration in response to oxygen-glucose deprivation in rat hippocampal slices. Neuroscience Letters 168, 155-158. Endres, W., Ballanyi, K. , Serve, G. and Grafe, P. (1986). Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord. Neuroscience Letters 72, 54-58. Felder, C. C , Albrecht, F. E., Campbell, T., Eisner, G. M . and Jose, P. A. (1993). cAMP-independent, G protein-linked inhibition of NaTET exchange in renal brush border by D[ dopamine agonists. American Journal of Physiology 264, F1032-F1037. Flagg-Newton, J. L. , Dahl, G. and Loewenstein, W. R. (1981). Cell junction and cyclic A M P : I. Upregulation of junctional membrane permeability and junctional membrane 160 particles by administration of cyclic nucleotide or phosphodiesterase inhibitor. Journal of Membrane Biology 63, 105-121. Foehring, R. C , Schwindt, P. C. and Crill, W. E. (1989). Norepinephrine selectively reduces slow Ca 2 + - and Na+-mediated K + currents in cat neocortical neurons. Journal of Neurophysiology 61, 245-256. Fowler, J. C. and O'Donnell, J. M . (1988). Antagonism of the responses to isoproterenol in the rat hippocampal slice with subtype-selective antagonists. European Journal of Pharmacology 153, 105-110. Frelin, C , Vigne, P., Ladoux, A . and Lazdunski, M . (1988). The regulation of the intracellular pH in cells from vertebrates. European Journal of Biochemistry 174, 3-14. Frey, G., Hanke, W. and Schlue, W.-R. (1993). ATP-dependent and Ca2+-dependent K + channels in the soma membrane of cultured leech retzius neurons. Journal of Membrane Biology 134, 131-142. Frotscher, M . , Kugler, P., Misgeld, U . and Zilles, K. (1990). Neurotransmission in the hippocampus. In. Advances in Anatomy, Embryology and Cell Biology 111, pp. 2-19. Eds. F. Beck, W. Hild, W. Kriz, R. Ortmann, J. E. Pauly, and T. H. Schiebler. Springer-Velag. Gaillard, S. and DuPont, J.-L. (1990). Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture. Journal of Physiology 425, 71-83. Gambassi, G., Spurgeon, H. A. , Lakatta, E. G , Blank, P. S. and Capogrossi, M . C. (1992). Different effects of a- and B-adrenergic stimulation on cytosolic pH and myofilament responsiveness to Ca 2 + in cardiac myocytes. Circulation Research 71, 870-882. Ganz, M . B . and Boron, W. F. (1994). Long-term effects of growth factors on pH and acid-base transport in rat glomerular mesangial cells. American Journal of Physiology 266, F576-F585. Ganz, M . B. , Pachter, J. A . and Barber, D. L. (1990). Multiple receptors coupled to adenylate cyclase regulate Na-H- exchange independent of cAMP. Journal of Biological Chemistry 265, 8989-8992. Gean, P.-W., Huang, C . - C , Lin, J.-H. and Tsai, J.-J. (1992). Sustained enhancement of N M D A receptor-mediated synaptic potential by isoproterenol in rat amygdalar slices. Brain Research 594, 331-334. 161 Gereau, R. W. and Conn, P. J. (1994a). Presynaptic enhancement of excitatory synaptic transmission by P-adrenergic receptor activation. Journal of Neurophysiology 72, 1438-1442. Gereau, R. W. and Conn, P. J. (1994b). A cyclic AMP-dependent form of associative synaptic plasticity induced by coactivation of P-adrenergic receptors in rat hippocampus. Journal of Neuroscience 14,3310-3318. Gereau, R. W., Winder, D. G. and Conn, P. J. (1995). Pharmacological differentiation of the effects of co-activation of p-adrenergic and metabotropic glutamate receptors in rat hippocampus. Neuroscience Letters 186, 119-122. Giacobino, J.-P. (1995). p3-adrenoceptor: A n update. European Journal of Endocrinology 132, 377-385. Gi l l , D. M . and Woolkalis, M . J. (1991). Cholera toxin-catalyzed [32P]ADP-ribosylation of proteins. Methods in Enzymology 195, 267. Gilman, A . G. (1984). G proteins and dual control of adenylate cyclase. Cell 36, 577-579. Goldman, S. A. , Pulsinelli, W. A. , Clarke, W. Y. , Kraig, R. P. and Plum, F. (1989). The effects of extracellular acidosis on neurons and glia in vitro. Journal of Cerebral Blood Flow and Metabolism 9, All-All. Gottfried, J. A . and Chesler, M . (1994). Endogenous FT modulation of N M D A receptor-mediated EPSCs revealed by carbonic anhydrase inhibition in rat hippocampus. Journal of Physiology 478.3, 373-378. Green, J. and Kleeman, C. (1992). Role of calcium and cAMP messenger systems in intercellular pH regulation of osteoblastic cells. American Journal of Physiology 262, C111-C121. Greengard, P., Jen, J., Nairn, A . C. and Stevens, C. F. (1991). Enhancement of the glutamate response by cAMP-dependent protein kinase in hippocampal neurons. Science 253, 1135-1138. Grinstein, S. and Rothstein, A . (1986). Mechanisms of regulation of the Na7H + exchanger. Journal of Membrane Biology 90,1-12. Grinstein, S., Rotin, D. and Mason, M . J. (1989). NaTET exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochimica et Biophysica Acta 988, 73-97. 162 Grynkiewicz, G., Poenie, M . and Tsien, R. Y . (1985). A new generation of Ca 2 + indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. Guizouarn, H . , Borgese, F., Pellissier, B. , Garcia-Romeu, F. and Motais, R. (1993). Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent NaTFT antiport. Journal of Biological Chemistry 268, 8632-8639. Guo, H. , Wasserstrom, J. A . and Rosenthal, J. E. (1992). Effect of catecholamines on intracellular pH in sheep cardiac Purkinje fibres. Journal of Physiology 458, 289-306. Gupta, A. , Schwiening, C. and Boron, W. F. (1994). Effects of CGRP, forskolin, P M A and ionomycin on pH ; dependence of Na-H exchange in UMR-106 cells. American Journal of Physiology 266, C1083-C1092. Haas, H. L . and Green, R. W. (1986). Effects of histamine on hippocampal pyramidal cells of the rat in vitro. Experimental Brain Research 62, 123-130. Haas, H . L . and Konnerth, A . (1983). Histamine and noradrenaline decrease calcium-activated potassium conductance in hippocampal pyramidal cells. Nature 302, 432-434. Harada, K. , Yoshimura, T. , Nakajima, K. , Ito, H. , Ebina, Y . and Shingai, R. (1992). N-methyl-D-asparate increases cytosolic Ca 2 + via G proteins in cultured hippocampal neurons. American Journal of Physiology 262, C870-C875. Harley, C. W. (1987). A role for norepinephrine in arousal, emotion and learning?: Limbic modulation by norepinephrine and the Kety hypothesis. Progress in Neuro-Psychopharmacology and Biological Psychiatry 11, 419-458. Harley, C. W., Lalies, M . D. and Nutt, D. J. (1996). Estimating the synaptic concentration of norepinephrine in dentate gyrus which produces P-receptor mediated long-lasting potentiation in vivo using microdialysis and intracerebroventricular norepinephrine. Brain Research 710, 293-298. Hartley, Z. and Dubinsky, J. M . (1993). Changes in intracellular pH associated with glutamate excitotoxicity. Journal of Neuroscience 13, 4690-4699. Heal, D. J . , Butler, S. A. , Prow, M . R. and Buckett, W. R. (1993). Quantification of presynaptic ot2-adrenoceptors in rat brain after short-term DSP-4 lesioning. European Journal of Pharmacology 249, 37-41. 163 Heginbotham, L. R. and Dunwiddie, T. V . (1991). Long-term increases in the evoked population spike in the CA1 region of the rat hippocampus induced by P-adrenergic receptor activation. Journal of Neuroscience 11, 2519-2527. Hidaka, H . and Koybayashi, R. (1992). Pharmacology of protein kinase inhibitors. Annual Review of Pharmacology and Toxicology 32, 377-397. Hille, B. (1992). G protein-coupled mechanisms and nervous signaling. Neuron 9, 187-195. Hopkins, W. F. and Johnston, D. (1988). Noradrenergic enhancement of long-term potentiation at mossy fiber synapses in the hippocampus. Journal of Neurophysiology 59, 667-687. Hortnagl, H , Berger, M . L. and Pifl, C. (1991). Regional heterogeneity in the distribution of neurotransmitter markers in the rat hippocampus. Neuroscience 45, 261-272. Huang, C.-C., Hsu, K.-S. and Gean, P.-W. (1996). Isoproterenol potentiates synaptic transmission primarily by enhancing presynaptic calcium influx via P- and/or Q-type calcium channels in the rat amygdala. Journal of Neuroscience 16, 1026-1033. Huang, C.-L., Cogan, M . G., Cragoe, E. J. and Ives, H . E. (1987). Thrombin activation of the Na7H + exchanger in vascular smooth muscle cells. Journal of Biological Chemistry 262, 14134-14140. Hughes, I. E. and Smith, J. A . (1978). The stability of noradrenaline in physiological saline solutions. Journal of Pharmacy and Pharmacology 30, 124-125. Humphreys, B. D., Jiang, L. , Chernova, M . N . and Alper, S. L . (1995). Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization. American Journal of Physiology 268, C201-C209. Hunter, T. (1995). Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 80, 225-236. Ikenouchi, H , Barry, W. H., Bridge, J. H. B., Weinberg, E. O., Apstein, C. S. Lorell, B. H. (1994). Effects of angiotensin II on intracellular Ca 2 + and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1. Journal of Physiology 480.2,203-215. Irisawa, H. and Sato, R. (1986). Intra- and Extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells. Circulation Research 59, 348-355. 164 Irwin, R. P., Lin, S.-Z., Long, R. T. and Paul, S. M . (1994). TV-methyl-D-aspartate induces a rapid, reversible and calcium-dependent intracellular acidosis in cultured fetal rat hippocampal neurons. Journal of Neuroscience 14, 1352-1357. Isom, L . L. , Cragoe, E. J. and Limbird, L . E. (1987). Multiple receptors linked to inhibition of adenylate cyclase accelerate N a + / H + exchange in neuroblastoma x glioma cells via a mechanism other than decreased cAMP accumulation. Journal of Biological Chemistry 262, 17504-17509. Ives, H. E. and Daniel, T. O. (1987). Interrelationship between growth factor-induced pH changes and intracellular Ca 2 + . Cell Biology 84, 1950-1954. Iwakura, K. , Hori, M . , Watanabe, Y . , Kitabatake, A. , Cragoe, E. J., Yoshida, H . and Kamada, T. (1990). aradrenoceptOr stimulation increases intracellular pH and Ca 2 + in cardiomyocytes through NaTFT and NaTCa 2 + exchange. European Journal of Pharmacology 186, 29-40. Jarolimek, W., Misgeld, U . and Lux, H. D. (1989). Activity dependent alkaline and acid transients in guinea pig hippocampal slices. Brain Research 505, 225-232. Jefferys, J. G. R. (1995). Nonsynaptic modulation of neuronal activity in the brain: Electric currents and extracellular ions. Physiological Reviews 75, 689-723. Johnson, R. A. and Shoshani, I. (1990). Kinetics of "P"-site-mediated inhibition of adenylyl cyclase and the requirements for substrate. Journal of Biological Chemistry 265, 11595-11600. Johnson, R. A . and Shoshani, I. (1994). Preparation and use of "P"-site-targeted affinity ligands for adenylyl cyclases. Methods in Enzymology 238, 56-58. Junitti-Berggren, L. , Ajkharnmar, P., Nilsson, T., Rorsman, P. and Berggren, P.-O. (1991). Glucose-induced increase in cytoplasmic pH in pancreatic p-cells is mediated by NaTFT exchange, an effect not dependent on protein kinase C. Journal of Biological Chemistry 266, 23537-23541. Kahn, A . M . , Bishara, M . , Cragoe, E. J., Allen, J. C , Seidel, C. L . Navran, S. S., O'Neil, R. G., McCarty, N . A . and Shelat, H . (1992). Effects of serotonin on intracellular pH and contraction in vascular smooth muscle. Circulation Research 71, 1294-1304. Kahn, A . M . , Dolson, G. M . , Hise, M . K., Bennett, S. C. and Weiman, E. J. (1985). Parathyroid hormone and dibutyryl cAMP inhibit NaTFT exchange in renal brush border vesicles. American Journal of Physiology 248, F212-F218. Kaibara, M . and Kameyama, M . (1988). Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea-pig. Journal of Physiology 403, 621-640. 165 Kaila, K. and Voipio, J. (1987). Postsynaptic fall in intracellular pH induced by G A B A -activated bicarbonate conductances. Nature 330, 163-165. Kaila, K. , Voipio, J., Paalasmaa, P., Pasternack, M . and Deisz, R. A . (1993). The role of bicarbonate in G A B A A receptor-mediated IPSPs of rat neocortical neurones. Journal of Physiology 464, 273-289. Kaku, D. A. , Giffard, R. G. and Choi, D. W. (1993). Neuroprotective effects of glutamate antagonists and extracellular acidity. Science 260, 1516-1518. Kandasamy, R. A. , Yu, F. H., Harris, R., Boucher, A. , Hanrahan, J. W. and Orlowski, J. (1995). Plasma membrane Na7H + exchanger isoforms (NHE-1, -2 and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. Journal of Biological Chemistry 270, 29209-29216. Kashiwagi, K. , Fukuchi, J.-I., Chao, J., Igarashi, K. and Williams, K . (1996). A n asparate residue in the extracellular loop of the jV-methyl-D-asparate receptor controls sensitivity to spermine and protons. Molecular Pharmacology 49, 1131-1141. Kettenmann, H . and Shlue, W.-R. (1988). Intracellular pH regulation in cultured mouse oligodendrocytes. Journal of Physiology 406, 147-162. Kikeri, D., Zeidel, M . L., Ballermann, B. J., Brenner, B. M . and Herbert, S. C. (1990). pH regulation and response to A V P in A10 cells differ markedly in the presence vs. absence of C 0 2 - H C 0 3 \ American Journal of Physiology 259, C471-C483. Kitamura, K. , Singer, W. D., Cano, A . and Miller, R. T. (1995). G a q and Grx13 regulate NHE-1 and intracellular calcium in epithelial cells. American Journal of Physiology 268,C101-C110. Koch, R. A . and 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. Journal of Neuroscience 14,2585-2593. Kohr, G. and Mody, I. (1991). Endogenous intracellular calcium buffering and the activation/inactivation of H V A calcium currents in rat dentate gyrus granule cells. Journal of General Physiology 98, 941-967. Kopito, R. R. (1990). Molecular biology of the anion exchanger gene family. International Review of Cytology 123, 177-199. Kopito, R. R., Lee, B. S., Simmons, D. M . , Lindsey, A . E., Morgans, C. W. and Schneider, K. (1989). Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59, 927-937. 166 Kraig, R. P., Petito, C. K. , Plum, F. and Pulsinelli, W. A. (1987). Hydrogen ions kill brain at concentrations reached in ischemia. Journal of Cerebral Blood Flow and Metabolism 7, 379-386. Kristian, T., Katsura, K. , Gido, G., Siesjo, B. K . (1994). The influence of pH on cellular calcium influx during ischemia. Brain Research 641, 295-302. Kume, H. , Takagi, K. , Satake, T., Tokuno, H. and Tomita, T. (1990). Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle. Journal of Physiology 424, 445-457. Lagadic-Gossman, D. and Vaughan-Jones, R. D. (1993). Coupling of dual acid extrusion in the guinea-pig isolated ventricular myocytes to c t r and B-adrenoceptors. Journal of Physiology 464, 49-73. Lahnsteiner, E. and Hermann, A . (1995). Acute action of ethanol on rat hippocampal CA1 neurons: Effects on intracellular signaling. Neuroscience Letters 191, 153-156. Laurenza, A. , Sutkowski, E. M . and Seamon, K. B. (1989). Forskolin: A specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action? Trends in Pharmacological Sciences Reviews 10, 442-447. Laurido, C , Candia, S., Wolff, D. and Latorre, R. (1991). Proton modulation of a C a 2 + -activated K + channel from rat skeletal muscle incorporated into planar bilayers. Journal of General Physiology 98, 1025-1043. Lee, S. C , Hamilton, J. S., Trammell, T., Horwitz, B. A . and Pappone, P. A . (1994). Adrenergic modulation of intracellular pH in isolated brown fat cells from hamster and rat. American Journal of Physiology 267, C349-C356. Limbird, L . E. (1988). Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms. FASEB Journal 2, 2686-2695. Loy, R., Koziell, D. A. , Lindsey, J. D. and Moore, R. Y . (1980). Noradrenergic innervation of the adult rat hippocampal formation. Journal of Comparative Neurology 189, 699-710. Ma, J. Y . , L i , M . , Caterall, W. A. and Scheuer, T. (1994). Modulation of brain Na + channels by a G protein-coupled pathway. Proceedings of the National Academy of Science, U.S.A. 91, 12351-12355. MacVicar, B. A . and Jahnsen, H. (1985). Uncoupling of CA3 pyramidal neurons by propionate. Brain Research 330, 141-145. 167 Madison, D. V . and Nicoll, R. A . (1986a). Actions of norepinephrine recorded intracellularly in rat hippocampal CA1 pyramidal neurones, in vitro. Journal of Physiology 372, 221-244. Madison, D. V . and Nicoll, R. A . (1986b). Cyclic 3', 5'-monophosphate mediates P-adrenergic actions of noradrenaline in rat hippocampal pyramidal cells. Journal of Physiology 372, 245-259. Martinez-Zaguilan, R., Gillies, R. J. and Sanchez-Armass, S. (1994). Regulation of pH in rat brain synaptosomes. II. Role of CI". Journal of Neurophysiology 71,2249-2257. Meech, R. W. (1979). Membrane potential oscillations in molluscan "burster" neurones. Journal of Experimental Biology 81, 93-112. Meech, R. W. and Mackie, G. O. (1993). Ionic currents in giant motor axons of the jellyfish, Aglantha digitate. Journal of Neurophysiology 69, 894-901. Minneman, K. P., Pittman, R. N . and Molinoff, P. B. (1981). P-adrenergic receptor subtypes: Properties, distribution and regulation. Annual Review of Neuroscience 4, 419-461. Mironov, S. L . (1995). Plasmalemmal and intracellular Ca 2 + pumps as main determinants of slow Ca 2 + buffering in rat hippocampal neurones. Neuropharmacology 34, 1123-1132. Mironov, S. L . and Lux, H. D. (1991). Cytoplasmic alkalinization increases high-threshold calcium current in chick dorsal root ganglion neurones. Pflugers Arch 419, 138-143. Moody, W. (1984). Effects of intracellular H + on the electrical properties of excitable cells. Annual Review of Neuroscience 7, 257-278. Moolenaar, W. H . (1986). Effects of growth factors on intracellular pH regulation. Annual Review of Physiology 48, 363-376. Moolenaar, W. H. , Tsien, R. Y . , van der Saag, P. T. and de Laat, S. W. (1983). Na7H + exchange and cytoplasmic pH in the action of growth factors in human fibroblasts. Nature 304, 645-648. Mori-Okamoto, J. and Tatsuno, J. (1988). Effects of noradrenaline on the responsiveness of cultured cerebellar neurons to excitatory amino acids. Brain Research 448, 259-271. Morimoto, Y . , Kemmotsu, O. and Morimoto, Y . (1994). Effect of lactic and C 0 2 acidosis on neuronal function following glucose-oxygen deprivation in rat hippocampal slices. Brain Research 654, 273-278. 168 Mueller, A . L. , Palmer, M . R., Hoffer, B. J. and Dunwiddie, T. V . (1982). Hippocampal noradrenergic responses in vivo and in vitro: Characterization of alpha and beta components. Naunyn-Schmiedeberg's Archives Pharmacology 318, 259-266. Munsch, T. and Deitmer, J. W. (1994). Sodium-bicarbonate cotransport in identified leech glial cells. Journal of Physiology 474.1, 43-53. Nachshen, D. A . and Drapeau, P. (1988). The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain. Journal of General Physiology 91, 289-303. Nedergaard, M . , Goldman, S. A. , Desai, S. and Pulsinelli, W. A . (1991). Acid-induced death in neurons and glia. Journal of Neuroscience 11, 2489-2497. Nicoll, R. A. , Malenka, R. C. and Kauer, J. A . (1990). Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiological Reviews 70, 513-565. Nowicky, A . V . , Christofi, G. and Bindman, L . J. (1992). Investigation of B-adrenergic modulation of synaptic transmission and postsynaptic induction of associative LTP in layer V neurone in slices of rat sensorimotor cortex. Neuroscience Letters 137, 270-274. O'Connor, E. R., Sontheimer, H. and Ransom, B. R. (1994). Rat hippocampal astrocytes exhibit electrogenic sodium-bicarbonate co-transport. Journal of Neurophysiology 72, 2580-2589. O'Donnell, S. R. and Wanstall, J. C. (1980). Evidence that ICI 118,551 is a potent, highly beta2-selective adrenoceptor antagonist and can be used to characterize beta-adrenoceptor populations in tissues. Life Sciences 27, 671-677. Orlowski, J., Kandasamy, R. A . and Shull, G. E. (1992). Molecular cloning of putative members of the Na/H exchanger gene family. Journal of Biological Chemistry 267, 9331-9339. Ou-yang, Y. , Kristian, T., Kristianova, V. , Mellergard, P. and Siesjo, B. K. (1995). The influence of calcium transients on intracellular pH in cortical neurons in primary culture. Brain Research 676, 307-313. Ou-yang, Y. , Mellergard, P. and Siesjo, B. K. (1993). Regulation of intracellular pH in single rat cortical neurons in vitro: A microspectrofluorometric study. Journal of Cerebral Blood Flow and Metabolism 13, 827-840. Owen N . E. (1986). Effect of catecholamines on Na/H exchange in vascular smooth muscle cells. Proceedings of the National Academy of Science, U.S.A. 91, 5431-5435. 169 Paalasmaa, P. and Kaila, K. (1996). Role of voltage-gated calcium channels in the generation of activity-induced extracellular pH transients in the rat hippocampal slice. Journal of Neurophysiology 75,2354-2360. Paalasmaa, P., Taira, T., Voipio, J. and Kaila, K. (1994). Extracellular alkaline transients mediated by glutamate receptors in the rat hippocampal slice are not due to a proton conductance. Journal of Neurophysiology 72, 2031-2033. Pappas, C. A. , Ullrich, N . and Sontheimer, H. (1994). Reduction of glial proliferation by K + channel blockers is mediated by changes in pEl. NeuroReport 6, 193-196. Parker-Bothelo, L. H. , Rothermel, J. D., Coombs, R. V . and Jastorff, B. (1988). cAMP analog antagonists of cAMP action. Methods in Enzymology, 159, 159-172. Peers, C. and Green, F. K. (1991). Inhibition of Ca2 +-activated K + currents by intracellular acidosis in isolated type I cells of the neonatal rat carotid body. Journal of Physiology 437, 589-602. Pietrobon, D., Prod'hom, B. and Hess, P. (1989). pH dependence of proton-induced current fluctuations with Cs +, K + and Na + as permeant ions. Journal of General Physiology 94, 1-21. Prod'hom, B., Pietrobon, D. and Hess, P. (1987). Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive C a 2 + channel. Nature 329, 243-246. Raley-Susman, K. M . , Cragoe, E. J., Sapolsky, R. M . and Kopito, R. R. (1991). Regulation of intracellular pH in cultured hippocampal neurones by an amiloride-insensitive N a + / H + exchanger. Journal of Biological Chemistry 266, 2739-2745. Raley-Susman, K. M . , Sapolsky, R. M . and Kopito, R. B . (1993). C17HCCV exchange function differs in adult and fetal rat hippocampal neurons. Brain Research 614, 308-314. Reuss, L . and Petersen, K . - U . (1985). Cyclic A M P inhibits Na7H + exchange at the apical membrane of Necturus gallbladder epithelium. Journal of General Physiology 85, 409-429. Rink, T. J., Tsien, R. Y . and Pozzan, T. (1982). Cytoplasmic pH and free M g 2 + in lymphocytes. Journal of Cell Biology 95, 189-196. Roos, A. and Boron, W. F. (1981). Intracellular pH. Physiological Reviews 61, 296-434. Sanchez-Armass, S., Martinez-Zaguilan, R. and Martinez, G. M . (1994). Regulation of pH in rat brain synaptosomes. I. Role of sodium, bicarbonate and potassium. Journal of Neurophysiology 71, 2236-2248. 170 Sardet, C , Fafournoux, P. and Pouyssegur, J. (1991). a-thrombin, epidermal growth factor and okadaic acid activate the N a + / H + exchanger, NHE-1, by phosphorylating a set of common sites. Journal of Biological Chemistry 266, 19166-19171. Saxena, R., Saksa, B . A . , Fields, A . P. and Ganz, M . B. (1993). Activation of Na/H exchanger in mesangial cells is associated with translocation of P K C isoforms. American Journal of Physiology 265, F53-F60. Schwiening, C. J. and Boron, W. F. (1994). Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na+-dependent Q--HCO3- exchange. Journal of Physiology 475.1, 59-67. Schwiening, C. J., Kennedy, H. J. and Thomas, R. C. (1993). Calcium-hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons. Proceedings of the Royal Society London B Series 253, 285-289. Seamon, K. B. and Daly, J. W. (1981). Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. Journal of Biological Chemistry 256, 9799-9801. Segal, M . (1981a). The action of norepinephrine in the rat hippocampus: Intracellular studies in the slice preparation. Brain Research 206, 107-128. Segal, M . (1981b). Histamine modulates reactivity of hippocampal CA3 neurons to afferent stimulation in vitro. Brain Research 213, 443-448. Segal, M . and Bloom, F. (1974). The action of norepinephrine in the rat hippocampus. I. Iontophoretic studies. Brain Research 72, 79-97. Segal, M . , Greenberger, V . and Hofstein, R. (1981). Cyclic AMP-generating systems in rat hippocampal slices. Brain Research 213, 351-364. Segal, M . , Markram, H. and Richter-Levin, G. (1991). Actions of norepinephrine in the rat hippocampus. Progress in Brain Research 88, 323-329. Semrad, C. E. and Chang, E. B. (1987). Calcium-mediated cyclic A M P inhibition of Na-H exchange in small intestine. American Journal of Physiology 252, C315-C322. Shen, H. , Chan, J., Kass, I. S. and Bergold, P. J. (1995). Transient acidosis induces delayed neurotoxicity in cultured hippocampal slices. Neuroscience Letters 185, 115-118. Siarey, R. J., Andreasen, M . and Lambert, J. D. C. (1995). Serotoninergic modulation of excitability in area CA1 of the in vitro rat hippocampus. Neuroscience Letters 199, 211-214. 171 Siesjo, B. K. (1985). Acid-base homeostasis in the brain: Physiology, chemistry and neurochemical pathology. In: Progress in Brain Research, Vol . 63, pp. 121-154. Eds. K. Kogure, K . -A . Hossman, B. K. Siesjo and F. A. Welsh. Elsevier. Siesjo, B. K., von Hanwehr, R., Nergelius, G., Nevander, G. and Ingvar, M . (1985). Extra- and Intracellular pH in the brain during seizures and in the recovery period following the arrest of seizure activity. Journal of Cerebral Blood Flow and Metabolism 5, 47-57. Simon, R. P., Niiro, M . and Gwinn, R. (1993). Brain acidosis induced by hypercarbic ventilation attenuates focal ischemic injury. Journal of Pharmacology and Experimental Therapeutics 267, 1428-1431. Smellie, F. W., Davis, C. W., Daly, J. W. and Wells, J. N . (1979). Alkylxanthines: Inhibition of adenosine-elicited accumulation of cyclic A M P in brain slices and of brain phosphodiesterase activity. Life Sciences 24, 2475-2482. Smith, S. E., Gottfried, J. A. , Chen, J. C. T. and Chesler, M . (1994). Calcium dependence of glutamate receptor-evoked alkaline shifts in hippocampus. NeuroReport 5, 2441-2445. Somjen, G. G. (1984). Acidification of interstitial fluid in hippocampal formation caused by seizures and by spreading depression. Brain Research 311, 186-188. Spray, D. C. and Bennett, M . V. L. (1985). Physiology and pharmacology of gap junctions. Annual Review of Physiology 47, 281-303. Stanton, P. K. and Sarvey, J. M . (1985). Blockade of norepinephrine-induced long-lasting potentiation in the hippocampal dentate gyrus by an inhibitor of protein synthesis. Brain Research 361, 276-283. Stanton, P. K. , Mody, I. and Heinemann, U . (1989). Down-regulation of norepinephrine sensitivity after induction of long-term neuronal plasticity (kindling) in the rat dentate gyrus. Brain Research 476, 367-372. Summers, R. J., Papaioannou, M . , Harris, S. and Evans, B. A . (1995). Expression of p3-adrenoceptor mRNA in rat brain. British Journal of Pharmacology 116, 2547-2548. Sundaram, U. , Knickelbein, R. G. and Dobbins, J. W. (1991). Mechanism of intestinal secretion. Effect of serotonin on rabbit ileal crypt and villus cells. Journal of Clinical Investigation 87, 743-746. Taira, T., Smirnov, S., Voipio, J. and Kaila, K. (1993). Intrinsic proton modulation of excitatory transmission in rat hippocampal slices. NeuroReport 4, 93-96. 172 Takahashi, K.-I., Dixon, D. B. and Copenhagen, D. R. (1993). Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina. Journal of General Physiology 101, 695-714. Tang, C. -M. , Dichter, M . and Morad, M . (1990). Modulation of JV-methyl-D-aspartate channel by extracellular H+. Proceedings of the National Academy of Science, U.S.A. 87, 6445-6449. Terzic, A. , Puceat, M . , Clement, O., Scamps, F. and Vassort, G. (1992). a radrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells. Journal of Physiology 447,275-292. Thalmann, R. H. (1988). Evidence that guanosine triphosphate (GTP)-binding proteins control a synaptic response in brain: Effect of pertussis toxin and GTPyS on the late inhibitory postsynaptic potential of hippocampal CA3 neurons. Journal of Neuroscience 8, 4589-4602. Thomas, J. A. , Buchsbaum, R. N . , Zimniak, A. and Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210-2218. Thomas, R. C. (1984). Experimental displacement of intracellular pH and the mechanism of its subsequent recovery. Journal of Physiology 354, 3P-22P. Thompson, W. J. (1991). Cyclic nucleotide phosphodiesterases: Pharmacology, biochemistry and function. Pharmacology and Therapeutics 51, 13-33. Tolkovsky, A . M . and Richards, C. D. (1987). N a + / H + exchange is the major mechanism of pH regulation in cultured sympathetic neurons: measurements in single cell bodies and neurites using a fluorescent pH indicator. Neuroscience 22, 1093-1102. Tombaugh, G. C. (1994). Mild acidosis delays hypoxic spreading depression and improves neuronal recovery in hippocampal slices. Journal of Neuroscience 14(9), 5635-5643. Tombaugh, G. C. and Somjen, G. C. (1996). Effects of extracellular pH on voltage-gated Na + , K + and Ca 2 + currents in isolated CA1 neurons. Journal of Physiology 493.3, 719-732. Traynelis, S. F. and Cull-Candy, S. G. (1990). Proton inhibition of 7V-methyl-D-aspartate receptors in cerebellar neurons. Nature 345, 347-350. Traynelis, S. F., Hartley, M . and Heinemann, S. F. (1995). Control of proton sensitivity of the N M D A receptor by RNA splicing and polyamines. Science 268, 873-876 173 Tyler, T. J. (1980). Brain slice preparation: Hippocampus. Brain Research Bulletin 5, 391-403. Ueda, F., Kameda, Y . , Yamamoto, O. and Shibata, Y . (1994). 2?eta-adrenergic regulation of gap-junctional intercellular communication in cultured rabbit gastric epithelial cells. Journal of Pharmacology and Therapeutics 271, 397-402. Umbach, J. A . (1982). Changes in intracellular pH affect calcium currents in Paramecium caudatum. Proceedings of the Royal Society of London B 216, 209-224. Velisek, L . , Dreier, J. P., Stanton, P. K., Heinemann, U . and Moshe, S. L . (1994). Lowering of extracellular pH suppresses low-Mg 2 + -induces seizures in combined entorhinal cortex-hippocampal slices. Experimental Brain Research 101, 44-52. Voipio, J., Paalasmaa, P., Taira, T. and Kaila, K. (1995). Pharmacological characterization of extracellular pH transients evoked by selective synaptic and exogenous activation of A M P A, N M D A and GAB A A receptors in the rat hippocampal slice. Journal of Neurophysiology 74, 633-642. von Hanwehr, R., Smith, M . L . and Siesjo, B. K. (1986). Extra- and intracellular pH during near-complete ischemia in the rat. Journal of Neurophysiology 46, 331-339. Voyno-Yasenetskaya, T., Conklin, B. R., Gilbert, R. L. , Hooley, R., Bourne, H . R. and Barber, D. L . (1994). G a 1 3 stimulates Na-H exchange. Journal of Biological Chemistry 269, 4721-4724. Vyklicky, L. , Vlachova, V . and Krusek, J. (1990). The effect of external pH changes on responses to excitatory amino acids in mouse hippocampal neurones. Journal of Physiology 430, 497-517. Wakabayashi, S., Sardet, C , Farfournoux, P., Counillon, L. , Meloche, S., Pages, G. and Pouyssegur, J. (1992). Structure function of the growth factor-activatable Na7H + exchanger (NHE1). Review in Physiology, Biochemistry and Pharmacology 119, 157-186. Wallert, M . A . and Frohlich, O. (1992). cc,-adrenergic stimulation of Na-H exchange in cardiac myocytes. American Journal of Physiology 263, C1096-C1102. Wang, G. J., Randall, R. D. and Thayer, S. A . (1994). Glutamate-induced intracellular acidification of cultured hippocampal neurons demonstrated altered energy metabolism resulting from Ca 2 + loads. Journal of Neurophysiology 72, 2563-2569. Wanke, E., Carbone, E. and Testa, P. L. (1979). K + conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane. Biophysical Journal 26, 319-324. 174 Weiman, E., Shenolikar, S. and Kahn, A. M . (1987). cAMP-associated inhibition of the Na + -H + exchanger in rabbit kidney brush-border membranes. American Journal of Physiology 252, F19-F25. Wera, S. and Hemmings, B. A . (1995). Serine/threonine protein phosphatases. Journal ofBiochemistry 311, 17-29. Wickman, K . and Clapham, D. E. (1995). Ion channel regulation by G proteins. Physiological Reviews 75, 865-885. Winkel, G. K. , Sardet, C , Pouyssegur, J. and Ives, H. E. (1993). Role of cytoplasmic domain of the Na7H + exchanger in hormonal activation. Journal of Biological Chemistry 268, 3396-3400. Wu, M . - L . and Tseng, Y . -Z . (1993). The modulatory effects of endothelin-1, carbachol and isoprenaline upon Na + -H + exchange in dog cardiac Purkinje fibres. Journal of Physiology 471, 583-597. Wu, M . - L . and Vaughan-Jones, R. D. (1994). Effect of metabolic inhibitors and second messengers upon Na + -H + exchange in the sheep cardiac Purkinje fibre. Journal of Physiology 478.2, 301-313. Wu, P. H. , Phillis, J. W. and Nye, M . J. (1982). Alkylxanthines as adenosine receptor antagonists and membrane phosphodiesterase inhibitors in central nervous tissue: Evaluation of structure-activity relationships. Life Sciences 31, 2857-2867. Yanovsky, Y . , Reyman, K . and Haas, H. L . (1995). pH-dependent facilitation of synaptic transmission by histamine in the CA1 region of mouse hippocampus. European Journal of Neuroscience 7, 2017-2020. Yuan, W. and Bers, D. (1995). Protein kinase inhibitor H-89 reverses forskolin stimulation of L-type calcium current. American Journal of Physiology 268, C651-C659. Yun, C. H. , Tse, C. -M. , Nath, S., Levine, S. L. and Donowitz, M . (1995). Structure/function studies of mammalian Na-H exchangers - An update. Journal of Physiology 482.P, \S-6S. Zilles, K. , Gross, G., Schildgen, S., Bauer, A. , Bahro, M . , Schwendemann, G., Zech, K. and Kolassa, N . (1991). Regional and laminar distributions of aradrenoceptors and their subtypes in human and rat hippocampus. Neuroscience 40, 307-320. Zorumski, C. F., Thio, L . L., Isenberg, K. E. and Clifford, D. B. (1992). Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons. Molecular Pharmacology 41, 931-936. 

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