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Anoxia-evoked changes in intracellular pH in acutely dissociated adult rat hippocampal neurons 1998

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ANOXIA-EVOKED CHANGES IN INTRACELLULAR pH IN ACUTELY DISSOCIATED ADULT RAT HIPPOCAMPAL NEURONS by CLAIRE ALEXIS SHELDON B.Sc. (Physiology), The University of Alberta, 1996 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE 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 December, 1998 © Claire Alexis Sheldon, 1998 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 k ^ j s i o ^ n ^ , The University of British Columbia Vancouver, Canada Date DE-6 (2/88) 11 A B S T R A C T The ratiometric fluorophore, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was utilized to measure pH; in acutely dissociated adult rat hippocampal CA1 neurons in response to periods of anoxia of 3, 5 and 10 min duration. At 37°C, in the presence or absence of HC0 3 " , the pH s response to anoxia was characterized by three phases: i) during the anoxic period, there was an intracellular acidification; ii) in the continued absence of oxygen, there was a rise in pHj toward pre-anoxic pHj values; and iii) in the immediate post-anoxic period, there was an intracellular alkalinization above pre-anoxic pHi values. These changes were not secondary to anoxia-evoked changes in intracellular free Ca concentration. r Next, the mechanisms underlying the anoxia-evoked changes in pH ; were examined. In addition to supporting the established roles of ,an increased rate of ATP hydrolysis and intracellular lactate accumulation, evidence is presented that a decreased activity of the Na7H + exchanger contributed to the production of the intracellular acidification observed during an anoxic period. The rise in pHj observed in the continued absence of oxygen was attributed, at least in part, to activation of a Zn2+-sensitive, voltage-dependent proton conductance. Finally, an increased activity of the Na7H + exchanger contributed to the production of the post-anoxic alkalinization. Reductions in extracellular pH and ambient temperature were found to attenuate the post-anoxic activation of Na7H + exchange, as was an inhibitor of P K A . The pHi changes characterized in the present study may determine, at least in part, neuronal viability following an anoxic insult. i i i TABLE OF CONTENTS Page Abstract i i Table of Contents i i i List of Tables vi List of Figures vii Acknowledgments x INTRODUCTION 1 Anoxia and Neuronal Viability 1 2 Neuronal Response to a Transient Anoxic Period 2 3 Mechanisms Contributing to Anoxia-Induced Neuronal Damage 4 3.1 Glutamate excitotoxicity 4 3.2 Role of extracellular pH 5 3.3 Role of intracellular pH 7 4 Intracellular pH Regulating Mechanisms 10 4.1 Na7H + exchange 11 4.2 HC03"-dependent pHj regulating mechanisms 14 4.3 Additional pHi regulating mechanisms 16 5 Summary 20 M A T E R I A L S A N D METHODS 1 Experimental Preparations 22 1.1 Acutely dissociated adult rat hippocampal C A I neurons 22 1.2 Cultured post-natal rat hippocampal neurons 23 2 Solutions and Chemicals 25 3 Microspectrofluorimetry 28 3.1 Cell loading and BCECF 28 3.2 Experimental setup and the ratiometric method 30 3.3 Calculation of pH; and [Ca 2 +] s 32 4 Experimental Procedures 34 4.1 Induction of anoxia 34 iv 4.2 Changes in steady-state pHj and rates of recovery of pH> 35 5 Analysis of Data 3 7 5.1 Analysis of steady-state changes in pHj 37 5.2 Analysis of the rates of recovery of pHj from imposed intracellular acid loads 3 9 RESULTS 1 Distribution of steady-state p r l under normoxic conditions 55 2 Characterization of the pH; response to anoxia 56 2.1 Steady-state changes in pH, evoked by anoxia under HEPES-buffered conditions 56 2.2 Steady-state changes in pH ; evoked by anoxia under H C 0 3 7 C 0 2 - buffered conditions 57 3 Changes in [Ca 2 +]; evoked by transient anoxia 5 9 4 Validation of the use of NajSjC^ to induce anoxia 59 5 Mechanisms underlying the pHi response to transient anoxia 6 2 5.1 Ro leo fCa 2 + 6 2 5.2 Role of the Na7H+exchanger 63 5.3 The effects of tetrodotoxin (TTX) 6 9 5.4 The potential involvement of a voltage-activated H + conductance 71 6 The effects of changing ambient temperature 7 2 6.1 Steady-state changes in pHj evoked by anoxia at 22°C under HEPES- buffered conditions , 7 2 6.2 Steady-state changes in pH, evoked by anoxia at 22°C under H C 0 3 - buffered conditions 73 6.3 Anoxia-evoked changes in the activity of the Na7H + exchanger at 22°C 74 7 The effects of changing extracellular pH 75 7.1 The effects of reducing extracellular pH 75 7.2 The effects of increasing extracellular pH 77 7.3 The dependence of intracellular pH on extracellular pH under normoxic conditions 79 8 The potential contribution of changes in intracellular buffering power 80 9 The role of extracellular glucose concentration 82 9.1 Role of the Na7H + exchanger at a reduced extracellular glucose concentration 84 V 10 The potential role of intracellular second messenger systems 86 11 pH- regulation in the absence of extracellular N a + 88 DISCUSSION 1 Intracellular pH regulation under normoxic conditions 165 1.1 Intracellular pH regulating mechanisms present in rat hippocampal C A I neurons 165 1.2 The role of extracellular pH in determining pHj 166 1.3 The role of ambient temperature in determining pHj 168 2 The intracellular pH response to a transient period of anoxia 169 2.1 Studies of the pHi response to anoxia in vivo and in slice preparations in vitro 170 2.2 The role of extracellular HC0 3 " in the production of the pH; response to anoxia 171 3 The intracellular C a 2 + response to a transient period of anoxia 172 3.1 Role of C a 2 + in the production of the pH; response to anoxia 174 4 Factors contributing to the anoxic acidification 175 4.1 Intracellular ATP during anoxia : 175 4.2 Intracellular lactate during anoxia 176 4.3 Changes in Na7H+exchange activity 177 4.4 Physiological relevance of the anoxic acidification 181 5 Factors contributing to the per-anoxic rise in intracellular pH 182 5.1 Role of a voltage-activated proton conductance 182 5.2 Role of lactate transport '. 185 6 Factors contributing to the post-anoxic alkalinization 186 6.1 Role of a voltage-activated proton conductance 187 6.2 Changes in Na7H + exchange activity 188 6.3 Potential physiological relevance of the post-anoxic activation of Na7Ff exchange 192 7 Summary 195 REFERENCES 201 vi LIST OF T A B L E S Page Table 1 Composition of HEPES-buffered experimental solutions 42 Table 2: Composition of HC037C02-buffered experimental solutions 43 Table 3 List of pharmacological agents employed 44 Table 4 Magnitude and time course of anoxia-evoked changes in intracellular pH under HC0 3-free, HEPES-buffered conditions 90 Table 5 Magnitude and time course of anoxia-evoked changes in intracellular pH under HC0 3VC0 2-buffered conditions 91 Table 6 The influence of the reduction of extracellular N a + on the pH; response to anoxia under HC03"-free, HEPES-buffered conditions 92 Table 7 Magnitude and time course of anoxia-evoked changes in pH; in the presence of T T X or ZnCl 2 93 Table 8 The influence of temperature on the magnitude and time course of anoxia-evoked changes in pH ; 94 Table 9 The influence of pH 0 on the magnitude and time course of anoxia- evoked changes in pHj 95 Table 10 Magnitude and time course of anoxia-evoked changes in pH ( under HC03"-free, HEPES-buffered conditions in the presence of 5.0 m M glucose 96 Table 11 The influence of the reduction of extracellular N a + on the anoxia- evoked changes in pH; under HC03"-free, HEPES-buffered conditions in the presence of 5.0 m M glucose 97 Table 12 Magnitude and time course of anoxia-evoked changes in pH, in the presence of agents that influence the activity of the c A M P / P K A pathway 98 Table 13 The influence of various pharmacological treatments on the recovery of pH, in the continued absence of extracellular Na +.... 99 vii LIST OF F I G U R E S Page Figure 1 Acutely dissociated adult rat hippocampal C A I neurons 45 Figure 2 Sample calibration plot for BCECF 47 Figure 3 Experimental parameters measured to assess the steady-state pH ; response to a transient period of anoxia 49 Figure 4 The pH ; response upon exposure to a reduced-[Na+]0 medium 51 Figure 5 Control acid load recoveries during perfusion with HC0 37C0 2-free, HEPES-buffered medium 53 Figure 6 Distributions of steady-state pHj values for all neurons during perfusion with HC0 37C0 2-free, HEPES-buffered and HC0 37C0 2-buffered media at37°C 100 Figure 7 Effects of a 5 min period of anoxia on steady-state pH, during perfusion with HC0 37C0 2-free, HEPES-buffered medium 102 Figure 8 Effects of a 3 and 10 min period of anoxia on steady-state pH ; during perfusion with HC0 37C0 2-free, HEPES-buffered medium 104 Figure 9 Effects of a 3, 5 and 10 min period of anoxia on steady-state pH ( during perfusion with HC0 37C0 2-buffered medium 106 Figure 10 Effects of a 5 min period of anoxia on BI334/BI3i0 ratio values 108 Figure 11 Effects of a 5 min exposure to HC0 37C0 2-buffered medium equilibrated with 95% Ar/ 5% C 0 2 for > 18 hours on steady-state pHj 110 2+ Figure 12 Effects of the removal of extracellular Ca on the pH; response to a 5 min period of anoxia 112 2+ 2+ Figure 13 Effects of the removal of extracellular Ca ontheCa response to a 5 min period of anoxia 114 Figure 14 Effects of exposure to reduced Na + , NMDG+-substituted medium on the pH| response to a 5 min period of anoxia 116 Figure 15 Effects of exposure to reduced Na + , Li+-substituted medium on the pH; response to a 5 min period of anoxia 118 vii i Figure 16 Effect of an anoxic period on the rate of pHj recovery from an intracellular acid load imposed during the anoxic period 120 Figure 17 Comparison of the rates of pHj recovery from intracellular acid loads imposed prior to and during an anoxic period 122 Figure 18 Effect of a transient anoxic period on the rate of pHj recovery from an intracellular acid load 124 Figure 19 Comparison of the rates of pH, recovery from intracellular acid loads imposed prior to and following 5 min periods of anoxia 126 Figure 20 Effects of 0.5 u M tetrodotoxin (TTX) on the steady-state pH; response to a 5 min period of anoxia 128 Figure 21 Effect of 0.5 u M TTX on the rate of pHj recovery from intracellular acid loads imposed prior to and during the anoxic period ; 130 Figure 22 Comparison of the rates of pH ; recovery from intracellular acid loads imposed prior to and during an anoxic period in the presence or absence of T T X 132 Figure 23 Effects of 500 u M ZnCl 2 on the steady-state pHj response to a 10 min period of anoxia 134 Figure 24 Distributions of steady-state pH ; values for all neurons during perfusion with HC0 37C0 2-free, HEPES-buffered and HC0 37C0 2-buffered media at22°C 136 Figure 25 Effects of a 5 min period of anoxia on steady-state pHj at 22°C 138 Figure 26 Effect of a transient period of anoxia on the rate of pHj recovery from an intracellular acid load at 22°C 140 Figure 27 Comparison of the rates of pH ; recovery from intracellular acid loads imposed prior to and following 5 min periods of anoxia at 22°C and 37°C '. 142 Figure 28 Effects of reducing pH 0 to 6.60 on the pH| response to a 5 min period of anoxia 144 Figure 29 Effect of a transient period of anoxia on the rate of pH ; recovery from an intracellular acid load imposed at pH 0 6.60 146 ix Figure 30 Comparison of the rates of pHj recovery from intracellular acid loads imposed prior to and following a 5 min period of anoxia at pH 0 6.60 and pH 07.35 148 Figure 31 Effects of increasing pH 0 to 7.60 on the pH; response to a 5 min period of anoxia 150 Figure 32 Effect of a transient anoxic period on the rate of pHi recovery from an intracellular acid load at pH 0 7.60 152 Figure 33 Comparison of the rates of pH ; recovery from intracellular acid loads imposed prior to and following a 5 min period of anoxia at pH 0 7.60 and pH 0 7.35 154 Figure 34 Relationship between pH 0and pHj in HC0 37C0 2-free, HEPES-buffered media under normoxic conditions 156 Figure 35 Effects of 3, 5 and 10 min periods of anoxia on steady-state pH; during perfusion with media containing 5.0 m M glucose 158 Figure 36 Effects of exposure to reduced N a + medium on the pH; response to a 5 min period of anoxia in media containing 5 m M glucose 160 Figure 37 Effects of 50 u M Rp-cAMPS and 10 u M noradrenaline on the pHj response to a 5 min period of anoxia 162 Figure 38 Effect of a 5 min period of anoxia on the [Na+]j 197 Figure 39 pH s regulating mechanisms that may contribute to the anoxia-evoked changes in pH, observed in acutely dissociated adult rat hippocampal C A I neurons in response to transient periods of anoxia 199 X A C K N O W L E D G M E N T S There are a number of people that I would like to acknowledge. First and foremost, I would like to thank Dr. John Church for his support and guidance. I have sincerely enjoyed working with John. He has been instrumental to my development as a scientist. I would also like to thank members of my supervisory and examining committees, Drs. Kenneth Baimbridge, Steven Kehl, Lynn Raymond, Raymond Pederson, Edwin Moore and Valdimir Palaty, for sharing both their time and scientific knowledge. I would also like to acknowledge all members of the Departments of Physiology and Anatomy (students and faculty alike) for providing an atmosphere of achievement and personal growth. I would like to thank Stella Atmadja and Joe Tay for their technical expertise. Finally, I would like to thank Candace Hofmann, Craig Kamimura, and Chris Brett for their support and understanding. I would like to thank my entire family for their never-ending love and encouragement. I am indebted to you all. I I N T R O D U C T I O N 1 Anoxia and Neuronal Viability Due to a lack of energy storage and a high metabolic rate, neurons within the central nervous system are extremely sensitive to oxygen and/or glucose deprivation. Several seconds following the induction of hypoxia and/or ischemia, there is a slowing of cortical electroencephalographic activity and a loss of consciousness and higher brain functions (Hansen, 1985; Haddad and Jiang, 1993; Fortuna et al, 1997). On a cellular level, a transient period of ischemia of as little as 2.5 - 5.0 minutes can cause severe neuronal damage to the hippocampal CA1 region, the substantia nigra, the parietal cortex and the thalamic nuclei (Diemer and Siemkowicz, 1981; L i et al, 1996; Fortuna et al, 1997). Histological examination reveals regions of extensive neuronal necrosis, with neurons exhibiting characteristically shrunken cytoplasm and nuclei (Diemer and Siemkowicz, 1981; Fortuna et al, 1997) surrounded by transition areas of incomplete ischemia which also contain cellular damage (penumbral zones; Choi, 1990). It has been suggested that it is the reduction in oxygen availability, and not the reduction in blood flow (and, therefore, glucose supply), which is the important determinant of the neuronal response to an anoxic/ischemic insult (Astrup et al, 1981). As the partial pressure of oxygen is reduced, progressing from an hypoxic to an anoxic insult, neuronal function is more severely compromised and there is a decreased likelihood of functional recovery upon reoxgenation (Siesjo et al, 1975; O'Reilly et al, 1995). Thus, the reduction in oxygen availability is the major factor which determines the neuronal response to a transient period of ischemia. 2 2 Neuronal Response to a Transient Anoxic Period In response to a transient period of anoxia, neurons undergo a progressive series > of intracellular events which determine their excitability and viability during and following the anoxic period. These changes, while more exaggerated and more rapid to their onset, also underlie the neuronal response to a transient ischemic period. Moreover, this neuronal response has been confirmed in hippocampal, brainstem, and neocortical neurons (Cowan and Martin, 1992; Donnelly et al, 1992; Jiang and Haddad, 1992; O'Reilly et al, 1995; Tanaka et al, 1997). The primary event during an ischemic or anoxic period is a compromised cellular energy metabolism. Immediately upon the induction of a period of energy imbalance, there is a rapid decline in phosphocreatine (PCr) levels, which acts to maintain intracellular ATP levels for a short period of time (~ 30 - 90 seconds; Whittingham et al, 1984; Katsura et al, 1992b). During this period, there are only minor changes in the cellular membrane potential, intracellular pH (pFL) and/or extracellular pH (pH 0; Martin et al, 1994). Once the PCr supply is depleted, ATP levels begin to fall along with parallel increases in ADP and A M P levels, which, in turn, stimulate the glycolytic rate- controlling enzyme, phosphofructokinase (Erecinska and Silver, 1989). As a result of an increased rate of anaerobic glycolysis, pH i ? and then pH 0 , begin to decrease (Dennis et al, 1991; Katsura et al, 1992b; Smith et al, 1993). Approximately 2 minutes following the onset of anoxia, a now significant decrease in tissue ATP levels initiates electrophysiological failure (Lipton and Whittingham, 1982), which is signaled by an initial, slow decrease in membrane potential (Santos et al, 1996). In some cases, prior to 3 membrane depolarization, a slight hyperpolarization, attributed to an activation of ATP- sensitive K + currents (IKATP) a n d Ca2+-dependent K + currents (IK(ca2V Leblond and Krnjevic, 1989; Jiang and Haddad, 1991; Yamamoto et al, 1997) may be observed (Cummins et al, 1991). With a further decline in intracellular ATP levels, neurons undergo anoxic depolarization and there is a rapid redistribution of all major intracellular ions (Silver and Erecinska, 1990; Donnelly et al, 1992; Balestrino, 1995). Anoxic depolarization coincides with a rapid increase in intracellular Na ([NaD, intracellular Ca 2 ([Ca 2 !) and intracellular CI" ([CT];), a decrease in intracellular K + ([K+] ;), a loss of cell volume regulation and a further reduction in intracellular ATP levels (Jiang and Haddad, 1991; Katsura et al, 1992b; Tanaka et al, 1997; Fowler and L i , 1998). As a result of the severe intracellular ATP depletion and membrane depolarization, anoxia and ischemia cause a significant release of glutamate and monoamine neurotransmitters (Santos et al, 1996). In the continued absence of oxygen, neither the cellular energy status nor the membrane potential are able to recover. If the insult is transient in nature, the recovery of ATP levels to ~ 30% of pre-anoxic levels is sufficient to fuel Na7K + ATPase activity and restore transmembrane ion gradients (Ekholm et al, 1993). However, the restoration of transmembrane ion gradients following a transient period anoxia/ischemia does not occur consistently and the normalization of extracellular glucose and/or oxygen levels do not necessarily result in the restoration of ion homeostasis and membrane repolarization (O'Reilly et al, 1995; Tanaka et al, 1997). In fact, in peripheral cell types, cellular damage is initiated during the reperfusion period (Currin et al, 1991; Harper et al, 1993; Maddaford and Pierce, 1997). 4 3 Mechanisms Contributing to Anoxia-Induced Neuronal Damage In mammalian central neurons, a great deal of experimental work has addressed the relationship between the cellular response to a transient period of anoxia or ischemia and the events that may mediate neuronal damage. 3.1 Glutamate excitotoxicity One theory proposed to explain anoxia/ischemia-induced neuronal damage centers around the concept of excitotoxicity. As a result of a significant membrane depolarization, an increase in [Na+] i5 [Ca 2 +] i ; and a decrease in intracellular ATP, anoxia/ischemia induces the release of excitatory neurotransmitters (most notably glutamate) into the synaptic cleft (Santos et al, 1996). The resulting excessive stimulation of 7Y-methyl-D-aspartate (NMDA) and possibly other subtype's of glutamate receptors causes a dramatic rise in [Ca2 +] i 3 which then initiates neuronal damage by aggravating the intracellular decline in ATP levels and stimulating a plethora of intracellular degradative enzymes (Choi, 1990; Herman et al, 1990; Mitani et al, 1994). However, a number of studies have suggested that glutamate-mediated excitotoxicity may not be a completely valid model for the detrimental effects of anoxia on neuronal viability. Firstly, while glutamate receptor antagonists are neuroprotective following a period of combined oxygen and glucose deprivation, they fail to influence neuronal survival following a period of severe oxygen deprivation (Newell et al, 1995). Secondly, the time course of glutamate-induced neuronal dysfunction is distinct from that induced by anoxia (Friedman and Haddad, 1993; Haddad and Jiang, 1993). Thirdly, an increase 5 in [Ca ]j does not necessarily proceed to neuronal death (Friedman and Haddad, 1993; L i et al, 1996). Finally, it is well-established not only that the N M D A receptor-channel complex is inhibited by reductions in pH 0 {e.g. Vyklicky et al, 1990) but also that marked falls in pH 0 occur during anoxia and/or ischemia in vivo {e.g. Silver and Erecinska, 1992). These facts have lead some investigators to question the contribution of N M D A receptor- mediated excitotoxicity to neuronal death evoked by anoxia/ischemia in vivo (Tombaugh and Sapolsky, 1993). 3.2 The role of extracellular pH Rapid shifts in pH 0 occur in response to both physiological and pathophysiological events (Hansen, 1985) and these shifts are able to modulate neuronal function under both normoxic and anoxic conditions. Increased neuronal activity causes an initial extracellular alkalinization often followed by an extracellular acidification (Jarolimek et al, 1989; Rose and Deitmer, 1995). Moreover, during such pathological events as cardiac arrest and spreading depression, cerebral pH 0 transiently declines followed by an alkaline shift that gives way to a prolonged acidosis (Mutch and Hansen, 1984; Chesler, 1990). These pH shifts observed in the cerebral interstitial space range in magnitude from 0.1 to 1.0 pH units (Hansen, 1985) and, in turn, pH 0 changes of corresponding magnitudes are able to influence neuronal function (Balestrino and Somjen, 1988; Church and McLennan, 1989; Tombaugh, 1994; Tombaugh and Somjen, 1996). For example, seizure activity induces a decrease in pH 0 which may, in turn, act to limit or even terminate the seizure activity 6 itself (e.g. Aram and Lodge, 1987). In a similar manner, mild extracellular acidosis, which occurs during and following an anoxic insult, exerts a neuroprotective effect, while mild extracellular alkalosis is detrimental to neuronal survival. The neuroprotective ability of mild extracellular acidosis has been largely attributed to the ability of extracellular protons to inhibit Ca2+-influx through both N M D A and voltage-activated Ca 2 + channels (Church and McLennan, 1989; Giffard et al, 1990; Traynelis and Cull- Candy, 1991; Tombaugh and Somjen, 1996; Church et dl, 1998). In addition, mild extracellular acidosis suppresses neuronal excitability and reduces neuronal energy demand by decreasing voltage-gated Na + and Ca 2 + currents and augmenting G A B A A - mediated CI" current (Krnjevic and Walz, 1990; Cummins et al, 1991; Robello et al, 1994; Tombaugh and Somjen, 1996). These observations indicate that reductions in pH„ may exert a neuroprotective effect which can exceed that produced by glutamate receptor antagonists alone, a possibility supported by direct experimental evidence (Tombaugh and Sapolsky, 1990; Kaku et al, 1993). In contrast to the neuroprotective effects of mild reductions in pH 0 , marked falls in pH 0 may kill neurons (reviewed by Siesjo et al, 1993; Tombaugh and Sapolsky, 1993; Siesjo et al, 1996; see also Nedergaard et al, 1991). Excessive and/or prolonged extracellular acidosis rapidly depletes tissue ATP levels and, following an anoxic insult, stimulates lipid peroxidation and protein denaturation as well as the accumulation of free radicals (Tombaugh and Sapolsky, 1993; Nedergaard and Goldman, 1993; Siesjo et al, 1996; Raley-Susman and Barnes, 1998). Thus, pH 0 is a key determinant of both neuronal function and viability during and following an ischemic/anoxic insult. 7 3.3 The role of intracellular pH The great majority of studies which have examined the effects of changes in pH 0 on neuronal function, whether under physiological or pathophysiological conditions, have not assessed the possible contribution of changes in pH, that may occur coincident with a change in pH 0 . In the large majority of peripheral cell types (e.g. myocytes, neutrophils, and cardiac Purkinje fibers), pH 0 has only a minor influence on pH, (the slope of linear regression line relating pH; to pH 0 , ApPIrApH,,, = 0.35 - 0.40; see Aickin, 1984; Wilding et al, 1992). In contrast, in cultured post-natal hippocampal neurons, neocortical neurons, and rat brain synaptosomes, pH 0 is a critical determinant of pHi (ApH^ApH,, = 0.75; Ou- Yang et al, 1993; Sanchez-Armass et al, 1994; Church et al, 1998). The influence of pH 0 on neuronal function under normoxic and anoxic conditions may then, at least in part, be secondary to a change in pH ; . Indeed, p l i is known to influence the activities of a large number of intracellular processes and the shifts in pHj which occur in response to an anoxic or ischemic insult have been suggested to be a critical determinant of the neuronal response to anoxia (see below). Thus, pH; regulates the activities of intracellular metabolic pathways and intracellular second messenger systems, both of which are critical to the genesis of anoxic/ischemic neuronal damage (Siesjo et al, 1996; Wieloch et al, 1996). The activity of phosphofructokinase, for example, is markedly pH; dependent with a change as small as 0.1 pH unit being able to completely activate or inactivate the glycolytic pathway (Busa and Nuccitelli, 1984). Similarly, adenylyl cyclase and cyclic nucleotide phosphodiesterase, those enzymes responsible for cAMP synthesis and hydrolysis, 8 respectively, are regulated by pHj such that an intracellular alkalinization causes an elevation in intracellular cAMP levels (Busa and Nuccitelli, 1984). Additional 2+ 2+ intracellular second messengers, including Ca , Ca /calmodulin (Busa and Nuccitelli, 1984), and inositol trisphosphate (Vignes et al, 1996), are similarly sensitive to changes in pH ; . In turn, both in vivo and in vitro studies have suggested that anoxia/ischemia- 2+ induced changes in membrane potential and [Ca ] i s for example, reflect, at least in part, a change in p l i (Silver and Erecinska, 1990 and 1992; Cowan and Martin, 1995). In addition, Krnjevic and Walz (1990) observed a strong correlation between anoxia- induced falls in pH 0 and the degree of depression of the population spike in the C A I region of hippocampal slices, and they attributed this effect on a concomitant fall in pH,. Reductions in p r l evoked by anoxia or ischemia have been suggested to exert a 2+ neuroprotective effect. Thus, reductions in pHj can act to inhibit Ca influx via voltage- 2+ activated Ca channels (Takahashi and Copenhagen, 1996; Tombaugh and Somjen, 1997); reduce neurotransmitter release (Nachshen and Drapeau, 1988); and reduce gap junctional conductance, thereby inhibiting neuronal synchrony and (possibly) anoxia- induced epileptiform activity (Spray et al, 1985; Church and Baimbridge, 1991; Perez- Velazquez et al, 1994). On the other hand, the neurotoxicity associated with exposure to highly acidic media (pH 0 < 6.5) appears to be a function of the degree and duration of the intracellular acidification produced consequent upon the fall in pH 0 (Nedergaard et al, 1991). Thus, a marked decrease in pHi is capable of inducing cellular dysfunction by initiating D N A damage and the production of free radicals (Siesjo et al, 1996), as well as stimulating cellular swelling (Jakubovicz and Klip, 1989), all of which have been 9 implicated in anoxia/ischemia-induced neuronal damage (Choi, 1990; Tombaugh and Sapolsky, 1993). The studies cited above indicate that changes in intracellular pH can modulate not only neuronal activity under normoxic conditions but also neuronal viability in response to excitotoxic, anoxic, or ischemic insults. Indeed, differences in the magnitudes of the pH; changes evoked by anoxia have been suggested to underlie the increased resistance of fetal neurons to anoxia-induced injury (Roberts, Jr. and Chih, 1997), the neurotoxic nature of pre-anoxic hyperglycemia compared with hypoglycemia (LeBlanc et al, 1993; Tyson et al, 1993), and the neuroprotective nature of mild hypothermia during and following an anoxic insult (Laptook et al, 1995a). Thus, the changes in pHj which occur following an anoxic or ischemic insult may participate in determining cellular viability. In cardiac myocytes and hepatocytes, for example, the return to physiological pHj values in the immediate post-anoxic period, rather than the fall in pH, which is observed during anoxia, initiates an intracellular cascade of events which results in cellular damage (Currin et al, 1991; Bond et al, 1993; Miyame et al, 1996). The relevance of the "pH paradox" has not been established in central neurons, although a similar sequence of events may occur, given the recent demonstration that neuronal damage in cultured fetal ./ neocortical neurons evoked by metabolic inhibition (a combination of 2-deoxy-D-glucose and cyanide) can be reduced by inhibiting the rate of restoration of pHj to normal values in the immediate post-anoxic period (Vornov et al, 1996). 10 4 Intracellular pH Regulating Mechanisms As the intracellular compartment is significantly more alkaline than predicted i f protons were passively distributed across the plasma membrane, mechanisms must be in place to extrude intracellular protons. However, compared to that of invertebrate neurons, there are relatively few studies investigating pHj regulatory mechanisms in vertebrate, and, in particular, mammalian central neurons, even under normoxic conditions (reviewed by Chesler, 1990). This is surprising given that the dysfunction of specific pHj regulating mechanisms has been linked to such pathophysiological disorders as slow-wave epilepsy (Cox et al, 1997), Alzheimer's dementia (Bosman et al, 1997), glutamate excitotoxicity (Hartley and Dubinsky, 1993) and, indeed, in peripheral cell types, anoxia/ischemia-induced cellular damage (Scholz and Albus, 1993; Bugge et al, 1996; Piper et al, 1996). Thus, given the importance of pHj in regulating neuronal excitability and viability, the characteristics of the pHj regulating mechanisms involved in maintaining pH s are of critical importance. To date, two major classes of pH ; regulatory mechanisms have been found to be present in the central nervous system: i) a HC03"-independent, Na7H + exchanger, and ii) HC03"-dependent exchangers. However, given the large number of additional mechanisms which have been found to participate in pHj regulation in invertebrate neurons and vertebrate non-neuronal cells, it is unlikely that the aforementioned exchangers are the only mechanisms acting to regulate pHj in mammalian central neurons. 11 4.1 Nct/H* exchange Under normoxic conditions at 37°C, the resting pHj of rat hippocampal CA1 neurons, rat neocortical neurons, and rat brain synaptosomes is largely dependent on the activity of the NaTff exchanger (Raley-Susman et al, 1991; Ou-Yang et al, 1993; Sanchez-Armass et al, 1994; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998; but see Schwiening and Boron, 1994). To date, six isoforms of the mammalian Na7H + exchanger (NHE1-6) have been cloned, each demonstrating varying tissue distributions with a notable scarcity in the brain (Sardet et al, 1989; Counillon and Pouyssegur, 1995; Yun et al, 1995; Wakabayashi et al, 1997; Numata et al, 1998). The primary function of Na7H + exchange in all cell types studied to date is to extrude acid that accumulates intracellularly due to metabolism and the large inward transmembrane gradient for protons. In addition, Na7H + exchange activity serves to regulate steady-state intracellular N a + (Frelin et al, 1984; Pike et al, 1993), and, in a variety of specialized cell types, N a + reabsorption across epithelia, as well as cellular growth and proliferation (reviewed by Mahnensmith and Aronson, 1985; Noel and Pouyssegur, 1995; Yun et al, 1995). Common properties of the Na7H + exchanger present in both peripheral cell types and mammalian central neurons are that this antiport extrudes one intracellular H + in exchange for one extracellular Na + . In addition, extracellular L i + is an effective external N a + substitute (Kinsella and Aronson, 1981; Raley-Susman et al, 1991). Thus, this system is electroneutral, although the reversal of NHE1 activity has been associated with an outward proton conductance (Demaureux et al, 1995; see Section 4.3.2). A n additional common property which, at least in part, underlies the ability of the Na7H + 12 exchanger to respond to intracellular and extracellular signals is the presence of an intracellular proton modifier site, which, when protonated, increases the affinity of the Na7H + exchanger for intracellular protons (Aronson et al, 1982). Thus, in all cell types, both the rapidity of NaVFf exchange and its sensitivity for substrates (i.e. intracellular protons) can be tightly controlled. In contrast to NHE1-4 and the NaTFT exchangers present in other mammalian central neurons and glial cells (see Vornov et al, 1996; Bevensee et al, 1997a; Pedersen et al, 1998), all of which can be pharmacologically inhibited by amiloride, amiloride analogues and benzoyl guanidinium compounds (Tse et al, 1993; Yun et al, 1995), the NaTFT exchanger present in hippocampal neurons is completely insensitive to these compounds with concentrations as large as 1 m M having no effect on exchange activity (Raley-Susman et al, 1991; Schwiening and Boron, 1994; Baxter and Church, 1996). Furthermore, hippocampal Na7H + exchange activity is regulated by a number of intracellular and extracellular factors. Thus, the activity of the Na7H + exchanger present in hippocampal neurons is stimulated by a decrease in pHj (Raley-Susman et al, 1991; Baxter and Church, 1996; Smith et al, 1998), activation of P-adrenoceptors and/or the cAMP/protein kinase A (PKA) pathway (Smith et al, 1998), an increase in ambient temperature (Baxter and Church, 1996), and in response to hyperosmotic cell shrinkage (Bookstein et al, 1996). Although it is established that noradrenaline and the c A M P / P K A second messenger pathway can modulate the activity of the Na7H + present in hippocampal neurons (Smith et al, 1998), the activity of the NaTFT exchanger present in a variety of peripheral cell types can be controlled by a wide variety of external agents 13 and intracellular second messenger systems. These agents are not active in all cell types and may differ between neuronal preparations. For example, in contrast to acutely dissociated hippocampal neurons, in rat brain synaptosomes, Na7H + exchange activity is not regulated by either P K A or P K C and is primarily influenced by the levels of 9+ intracellular Ca (Sanchez-Armass et al, 1994). In addition, the intracellular pathways involved in the control of the activity of Na7H + exchangers in peripheral cell types are not mutually exclusive and the activity of a given Na7H + exchanger in a given cell type may be modulated concomitantly by more than one intracellular signaling pathway. It therefore appears likely that the activity of the Na7H + exchanger in rat hippocampal neurons may be controlled by pathways other than the c A M P / P K A pathway. Therefore, there is a distinct variant of the Na7H + exchanger present in hippocampal CA1 neurons. In recent years, isoforms NHE4 and NHE6 have been localized to the rat brain hippocampus and human brain tissue, respectively (Bookstein et al, 1996; Chambrey et al, 1997a; Numata et al, 1998). While NHE4 demonstrates a partial sensitivity to amiloride (K d = 0.8 mM; Chambrey et al, 1997b), NHE6 has been localized to the mitochondrial inner membrane and, as a result, its role in cytosolic pHi regulation is unclear. As a result, it is unlikely that either isoform represents the Na7H + exchange activity observed by our laboratory (Baxter and Church, 1996; Smith et al, 1998) and the laboratory of others (Raley-Susman et al, 1991; Bevensee et al, 1996). In parallel to its role under normoxic conditions, during and following either cardiac or cerebral ischemia, the Na7H + exchanger has been implicated in the regulation of pH ; , [Na+]i, and intracellular volume (Jakubovicz and Klip, 1989; Friedman and 14 Haddad, 1993; Askenasy et al, 1996). In fact, just as pH; is a key factor in determining cellular viability (see Section 3.3), overactivity of the Na7H + exchanger appears to be detrimental to functional recovery and survival following an anoxic or ischemic insult, although this relationship has been supported primarily by data collected in peripheral cell types, such as cardiac myocytes and hepatocytes (reviewed by Scholz and Albus, 1993; Piper et al, 1996; Bugge et al, 1996). Recently, in cortical neurons, Vornov et al (1996), demonstrated that Na7H + exchange inhibition, which blocked the pH, recovery following a period of metabolic inhibition, improved neuronal survival. Furthermore, given the facts that N a + / H + exchange activity may contribute to anoxia-evoked increase in [Na+]j (Friedman and Haddad, 1994b; Gleitz et al, 1996) and that an increase in [Na+]i may be involved in anoxia-evoked neuronal damage (Friedman and Haddad, 1994b; Taylor et al, 1995), it becomes increasingly possible that changes in the activity of the Na7H + exchanger may be related to subsequent neuronal viability following a transient anoxic insult. However although anoxia is known to evoke changes in pHj in vivo, the role of changes in the activity of the neuronal Na7H + exchanger in the production of these changes is unknown. 4.2 HCO{-dependent pH, regulating mechanisms In both peripheral cell types and central neurons, Na7H + exchange often co-exists with HC03"-dependent pH; regulatory mechanisms (Chesler, 1987; Boyarsky et al, 1988; Raley-Susman et al, 1991; Ou-Yang et al, 1993; Raley-Susman et al, 1993; Schweining and Boron, 1994; Baxter and Church, 1996; Smith et al, 1998). In hippocampal neurons, 15 two HC03'-dependent pHj regulatory mechanisms have been identified to date: i) a Na + - dependent HC0 3 7CT antiporter, which transports HC0 3 " into the cell in exchange for external CI", thereby functioning as an acid extruder; and ii) a Na+-independent HC037C1" antiporter which transports HC0 3 " out of the cell at physiological pHj values, thereby acting as an alkali extruder. This latter mechanism can reverse under conditions of extreme intracellular acidosis to participate in acid extrusion (Baxter and Church, 1996). Both HCO3VCI" exchangers are sensitive to inhibition by 4,4'-diisothiocyanatostilbene- 2,2'-disulfonate (DIDS). The regulation of the activity of HCO3VCI" exchangers in hippocampal neurons has not been investigated. However, in a manner similar to the Na7H + exchanger, H C 0 3 " /CI" exchangers in peripheral cell types respond to a variety of intracellular and extracellular factors, such as changes in pH 0 and the activities of various intracellular second messenger systems (Boron et al, 1979; Vigne et al, 1988; Ludt et al, 1991). Importantly, Na7H + exchange and HC03"/C1" exchange activity are often independently regulated. In osteoblasts, for example, a rise in [Ca ]; stimulates Na -independent H C 0 3 7 C r exchange but has no effect on Na7H + exchange activity whereas a rise in intracellular cAMP inhibits both exchangers (Green and Kleeman, 1992). Furthermore, while the mineralocorticoid, aldosterone, stimulates both Na7H + exchange and Na + - independent HC037C1" exchange in cardiac myocytes, the relative pattern of stimulation differs during cellular development (Korichneva et al, 1995). Similar to the Na7H + exchanger, changes in the activities of HC0 37C1" exchangers may play a role in the neuronal response to anoxia/ischemia. Thus, on the basis of NMR-based pH ( 16 measurements in hippocampal slices, Pirttila and Kauppinen (1994a) suggested that anoxia causes changes in the activities of HC03"-dependent pH ( regulatory mechanisms, although this study was unable to differentiate between the possible role of neuronal vs. glial elements in the effects observed. In addition, inhibition of HC0 37C1" exchange protects against excitotoxic damage in chick retinal cells (Zeevalk et al, 1989). Furthermore, in some cell types, forward Na+-independent HC0 3 7CT exchange participates in the maintenance of [CT]i and, thus, a particular value of the CI" equilibrium potential (e.g. Aickin and Brading, 1984; Vaughan-Jones, 1979). Given the importance of [H+] i 5 [HC03"]i, and [C\\ for the control of neuronal excitability (e.g. Kaila, 1994; Staley et al, 1995), it is possible (though untested) that HC0 37C1" exchange may be able to modulate excitability in mammalian central neurons. Thus, it is becoming increasingly recognized that, independent of NaTFT exchange, HC0 37C1" exchange regulates both pHj and neuronal excitability under a variety of physiological and pathophysiological conditions. 4.3 Additional pH, regulating mechanisms In the majority of cell types, a large number of mechanisms act in concert to regulate pHj. Under conditions which inhibit both NaTFT exchange and HC0 37C1" exchange, the demonstration of pHj regulation (i.e. recovery from an imposed intracellular acid load) supports the presence of alternative pHi regulating mechanisms in a variety of cell types (see Peral et al, 1995; Bevensee et al, 1996; Gibb et al, 1997). Outlined below are several significant pHj regulating mechanisms that can function in the 17 absence of Na7H + exchange and HC0 3 7Ci" exchange to regulate pHj under normoxic conditions and which may participate in the production of pHj changes during anoxia/ischemia. 4.3.1 Ca2+/H+ exchange There is a close, interdependent relationship between pI-L and [Ca ] v In addition to H and Ca ions competing for common intracellular binding sites, pHj regulates the 2+ influx of extracellular Ca through voltage-gated channels (Tombaugh and Somjen, 2+ 1997), the release of Ca from intracellular stores (Ou-Yang et al, 1994b), and the 2+ affinity of intracellular binding proteins (e.g. calmodulin) for Ca (Busa and Nuccitelli, 2+ 1984). In turn, Ca , both directly and via intracellular second messenger cascades, can regulate the activity of pHj regulating mechanisms (see Vaughan-Jones and Wu, 1990; Sanchez-Armass et al, 1994) in addition to influencing proton flux through voltage- dependent H + channels (Gordienko et al, 1996). Given the relationship between H + and 2+ Ca ions, it is perhaps not surprising that mechanism(s) exist to regulate in concert the internal concentrations of both ions. One such mechanism is the Ca 2 7H + ATPase, 2+ initially described in snail neurons which extrudes one intracellular Ca ion in exchange for an extracellular H + (Schweining et al, 1993). This mechanism may also exist in rat hippocampal neurons (Trapp et al, 1996). Ischemia or anoxia may affect the activity of 2+ this ATPase, given the facts that these insults lead a rise in [Ca ] i 5 and an external 2+ acidosis, both of which act to enhance to activity of the Ca / H + ATPase (see Schweining et al, 1993; Ou-Yang et al, 1994b). Others have suggested the existence of a reversible 18 C a 2 + / H + exchange mechanism which is not directly ATP-dependent (Ou-Yang et al, 1994a). 4.3.2 Voltage-activated proton conductance In a variety of cell types, a voltage-activated proton conductance (gH+) contributes to the recovery of pHj following intracellular acid loads imposed during membrane depolarization (Meech and Thomas, 1987; Byerly and Suen, 1989; Kapus et al, 1993; Gordienko et al, 1996). gu+s demonstrate an extremely high selectivity for H + and, upon activation, are capable of producing dramatic shifts in pH; (~ 5.0 pH units min"1), without demonstrating rapid current inactivation or desensitization (Meech and Thomas, 1987; Kapus et al, 1993; Lukacs et al, 1993; DeCoursey and Cherny, 1994). Although the existence of gH+ in mammalian central neurons has not been investigated, it is possible that it could be an important mechanism of prl, regulation during ischemic or anoxic insults which are associated with a prolonged membrane depolarization. In addition, evidence indicates that gH+s can couple to NaTFT exchange (Demaurex et al, 1995) such that activation of the electroneutral NaTFT exchanger can reduce the transmembrane H + current. 4.3.3 Proton ATPases P-type and V-type ATPases represent an integrated family of ATP-driven cation transporters. P-type ATPases are primarily found in the stomach, colon, and kidney, where they contribute to pH; regulation and K + homeostasis (van Driel and Callaghan, 1995). The V-type ATPase has been characterized in cardiac myocytes, renal epithelia and osteoclasts (Kurtz, 1987; Nelson and Klionsky, 1996). During and following periods 19 of metabolic inhibition, V-type ATPase activity provides a cardioprotective effect by attenuating the production of intracellular acidosis during the metabolic insult as well as 2+ decreasing Na7H + exchange-induced Ca overload (Karwatowska-Prokopcauk et al, 1998). 4.3.4 Lactate/lT cotransport Although neurons are primarily metabolically aerobic, during periods of intense neuronal activity or periods of ATP depletion, anaerobic metabolism becomes the key energy-producing pathway and, as a result, lactic acid accumulates intracellularly (Ljunggren et al, 191 A; Hope et al, 1988; Jarolimek et al, 1989). However, given a pK a of ~ 3.9, at physiological pH, values, lactic acid will exist primarily in its undissociated form and, thus, be unable to cross the lipid membrane (but see Dringen et al, 1995). In both peripheral cell types and mammalian central neurons, a lactate/H+ cotransport mechanism removes intracellular lactate and, in this way, may contribute to pHj regulation under normoxic conditions (Assaf et al, 1990; Nedergaard and Goldman, 1993; Pirttila and Kauppinen, 1994b; Juel, 1997). While some studies in peripheral cell types support a role for this mechanism in contributing to the recovery of pHj following transient periods of anoxia (Vandenberg et al, 1993), studies in mammalian central neurons have failed to document a similar role (Pirttila and Kauppinen, 1994b). 4.3.5 K+-dependent transporters A fifth class of pH; regulatory mechanisms are those that are K+-dependent, namely the K 7 H + exchanger and K7HC0 3 " exchanger (Hofer and Machen, 1992; Hogan et al, 1995a and 1995b; Zhao et al, 1995). These mechanisms have been shown to 20 participate in the recovery of pH( from imposed intracellular alkali or acid loads in squid giant axons (Hogan et al, 1995a and 1995b). Although these transporters have not been identified in mammalian central neurons, they may participate in pli regulation in hippocampal neurons and, furthermore, their role may become increasingly important in pH; regulation during periods of intracellular acidosis and/or a change in [K+]0 (e.g. membrane depolarization, periods of anoxia/ischemia). 5 Summary Although anoxia and ischemia have long been known to elicit changes in pH 0 and pH; both in vivo and in slice preparations in vitro (e.g. Fujiwara et al, 1992; Pirttila and Kauppinen, 1992, 1994a, 1994b; Melzian et al, 1996; Siesjo et al, 1996), it is difficult to separate the contribution of various cell types (including glia) to the effects observed under these experimental conditions. In addition, although changes in neuronal pH, associated with glutamate receptor-mediated excitotoxicity and metabolic inhibition have been described (e.g. Hartley and Dubinsky, 1993; Vornov et al, 1996), these insults are not a completely valid model for the direct action of anoxia on central neurons (see Friedman and Haddad, 1993). Furthermore, the mechanism(s) responsible for the pHj shifts observed in response to either anoxia, ischemia or excitotoxin exposure have not been rigorously characterized. Recently, data concerning the mechanisms which participate in pH; regulation in mammalian central neurons under normoxic conditions have become available and support a role for both Na7H+ exchange and HC037C1" exchange. However, there is 21 precedence for alternative pHj regulatory mechanisms which may function, as they do in peripheral cell types, to regulate pH, during both normoxic and anoxic conditions. Because changes in pHj may play an important role in the pathophysiology of neuronal death following anoxia and/or ischemia, a knowledge of the pH, changes evoked by anoxia in central neurons and the mechanisms involved in the regulation of neuronal pHj both during and following anoxia may provide insights into the pathogenesis of neurodegenerative phenomena. The aim of the present study, therefore, is to characterize the pH; changes which occur in response to transient anoxia in acutely dissociated adult rat CA1 hippocampal neurons, an experimental preparation in which the pH, regulating mechanisms operating under normoxic conditions have been extensively characterized (Schweining and Boron, 1994; Bevensee et al, 1996, Smith et al, 1998) and in which changes in pHj can be reliably quantified and their underlying mechanisms investigated. 22 MATERIALS AND METHODS 1 Experimental Preparations 1.1 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 (180 - 200 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). Food (Lab Diet, PMI Feeds Inc., St. Louis, MO) and water were available ad libitum. The animals were anesthetized with 3% halothane in air, 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 described by Tyler (1980) and transverse hippocampal slices, 450 pm thick, were obtained with a Mcllwain tissue chopper and collected in ice-cold (4°C - 8°C) HC0 37C0 2-buffered media. The slices were then transferred to an incubation chamber containing HC0 37C0 2-buffered saline and allowed to recover for at least one hour. Three hippocampal slices were then enzymatically digested at 32°C in 2.0 ml of HC0 3/C0 2-buffered saline containing 1.5 mg ml"1 of pronase (protease type XIV bacterial from Streptomyces griseus; Sigma Chemical Co., St. Louis, MO). After 30 min, the CA1 region of each slice was then microdissected with a dissecting chisel and triturated with fire-polished Pasteur pipettes of diminishing 23 tip diameters (0.7, 0.5, 0.3, and 0.2 mm) in 0.5 ml of loading medium, consisting of HEPES-buffered saline (Table 1, solution 1), pH 7.35 at room temperature, with the iso- osmotic addition of 3.0 m M NaHC0 3 replacing NaCl. The triturated mixture was then deposited onto an 18 mm poly-D-lysine- (20-40 ug ml"1; Sigma Chemical Co.) coated glass coverslip. The neurons were allowed to adhere to the substrate for 15 min at room temperature exposed to a 100% 0 2 atmosphere prior to the start of an experiment. Freshly isolated hippocampal C A I neurons were chosen for study based on morphological criteria established by Schwiening and Boron (1994), i.e. a smooth, non- granular appearance; a single major process (presumably an apical dendrite) projecting from one pole of the soma which was at least three times the length of the diameter of the cell body; and the presence of two or more smaller processes (basal dendrites) at the opposite pole (Fig. 1). 1.2 Cultured post-natal rat hippocampal neurons Cultured post-natal rat hippocampal neurons were employed only to derive parameters for the calibration of the BCECF signal (see Section 3.3). We have previously determined that the parameters derived from in situ calibration experiments using cultured neurons are identical to those obtained from in situ calibrations using acutely dissociated cells. Cultured post-natal rat 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; 24 Gibco, Grand Island, N Y ) and then incubated for 30 min at 37°C in L-15 medium containing 0.2 mg ml"1 bovine serum albumin, 1.0 mg ml"1 papain (type IV; Sigma Chemical Co.) and 25 ug ml"1 DNAase (type II; Sigma Chemical Co.). Afterwards, the L-15 medium was discarded and replaced with a medium containing Eagles's minimum essential medium ( E M E M ; Gibco), 22 m M NaHC0 3 , 10 m M 4-(2-hydroxyefhyl)-l- piperazineethanesulphonic acid (HEPES), 17 m M glucose, 400 u M glutamine and 25 ug ml"1 DNAase dissolved in tissue culture grade water. The slices were mechanically dissociated immediately with fire-polished Pasteur pipettes of decreasing tip diameters, underlay ed 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 cell pellet was re-suspended in E M E M . To determine 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"1. The cell suspension was then dispersed onto 18 mm glass 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 incubated in 5/5 serum E M E M for 24 h in a 5% C 0 2 atmosphere at 37°C. The growth medium was then switched to a serum-free, N2- supplemented E M E M (containing 5 ug ml"1 insulin, 20 n M progesterone, 100 u M putrescine and 30 nM sodium selenite) and the cell cultures were treated with 10 u M 25 cytosine-p-D-arabinofuranoside hydrochloride to suppress glial cell proliferation. The serum-free, N2-supplemented E M E M was half-changed with fresh medium every 3 - 4 days. Each coverslip consisted primarily of hippocampal C A I and CA3 neurons with a maximum of 15% of cells being glial. The cultured neurons were used in experiments from 4 -10 days after plating. A l l neuronal cultures were generously provided by Ms. Stella Atmadja, Department of Physiology, University of British Columbia. 2 Solutions and Chemicals Unless otherwise noted, all experiments were performed at 37°C. The HEPES- buffered and HC0 3/C0 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 Table 1 except for the concentration of glucose which was reduced to 10.0 m M from 17.5 m M . The reduced Na + , HEPES-buffered medium in which L i + was substituted for N a + was prepared by replacement of 134.5 m M NaCl and 1.5 mM NaH 2 P0 4 (Table 1, solution 1) with 136 m M L i C l . A l l low glucose solutions were prepared by reducing the concentration of glucose from 17.5 m M to 5.0 m M with the addition of 12.5 m M D- mannitol to maintain osmolarity (see Cendoroglo et al, 1998; Wang et al, 1998). The omolarities of these solutions were measured with an uOsmette osmometer (Precision Systems, Inc., Natick, M A ) , calibrated daily before use. 26 A l l solutions were prepared at room temperature (22°C). Accordingly, HEPES- buffered solutions were titrated to pH 7.48 - 7.50 (at room temperature) in order to achieve a final pH of 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 where pH 3 7 denotes the final pH of the HEPES-buffered saline at 37°C and pH 2 2 represents the pH to which the HEPES-buffered medium was titrated at room temperature (Baxter, 1995). To vary the pH of HC037C02-buffered solutions, the concentration of N a H C 0 3 was adjusted by equimolar substitution for NaCl (at a constant Pco2) according to the equation where pH 3 7 represents the pH of a HC0 3/C0 2-buffered solution at 37°C following equilibration with 5% C 0 2 (Baxter, 1995). The pH dependence of HC0 37C0 2-buffered media varies with temperature. For experiments performed at 22°C (room temperature), the appropriate concentration of NaHC0 3 was calculated according to the equation pH 3 7 = 0.1809 + (0.9673 x pH 2 2), (Equation 1) pH 3 7 = 6.017 + (1.036 x log[HC03-]) (Equation 2) pH 2 2 = 5.833 + (1.038 x log[HC0 3]) (Equation 3) 27 A l l HEPES-buffered solutions were titrated to the appropriate temperature- corrected pH value with 10 M NaOH, except for the high-K+ calibration medium (Table 1, solution 5) and the reduced-Na+, HEPES-buffered medium substituted with N M D G + (Table 1, solution 2) or L i + , which were titrated with 10 M K O H , 1.0 M HCI and 2.0 M L i O H , respectively. A Corning 240 pH meter (Corning Inc., Corning, N Y ) , calibrated daily, was utilized to measure the pH of all solutions. A list of pharmacological agents used in these studies is presented in Table 3. Unless otherwise noted, all agents were supplied by Sigma Chemical Co. Bafilomycin A] was a generous gift from Dr. V . Palaty (Department of Anatomy, University of British Columbia). Table 3 provides the stock concentrations and the solvent used to prepare the stock solution for each compound. Where applicable, the storage temperature is listed; however, i f not provided, the compound was prepared fresh immediately prior to an experiment. The final concentration of dimethylsulphoxide (DMSO) in the test solutions never exceeded 0.1%, which, in control experiments, fails to influence either steady-state pH, or the activities of the pH; regulating mechanisms in rat hippocampal neurons (Baxter and Church, 1996; Smith et al, 1998). Stock solutions were diluted to the appropriate test concentration in physiological saline. Solutions containing 500 u M ZnCl 2 did not contain N a H 2 P 0 4 , and M g C l 2 iso-osmotically replaced M g S 0 4 . 28 3.0 Microspectrofluorimetry 3.1 Cell loading and BCECF The microspectrofluorimetric technique used to measure pHj employed the intracellular fluorescent hydrogen ion indicator, 2',7'-bis-(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF; Molecular Probes Inc., Eugene, OR; Rink et al, 1982; Nett and Deitmer, 1996). The acetoxymethyl ester of BCECF (BCECF-AM) was prepared initially as a 1.0 m M stock solution in anhydrous dimethylsulphoxide (DMSO) and stored in 60 u,L aliquots at -60°C. B C E C F - A M is hydrophobic and uncharged, allowing it to permeate through the plasma membrane; however, upon entry into the neurons, BCECF- A M is hydrolyzed by intracellular esterases to produce the hydrophilic, polyanionic B C E C F free acid which becomes trapped within the neuron. For in situ calibration experiments employing cultured post-natal hippocampal neurons, a loading medium was prepared consisting of HEPES-buffered saline, pH 7.35 at room temperature (Table 1, solution 1), with the iso-osmotic addition of 3.0 m M N a H C 0 3 replacing NaCl. When needed, 5.0 pi of the 1.0 m M B C E C F - A M stock solution was added to 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 an O-ring rimmed with high vacuum silicone grease (Dow Corning, Midland, MI) so as to form the floor of the chamber. 29 For each experiment involving acutely dissociated adult hippocampal C A I neurons in which pH; was measured, 1.0 pi of 1.0 m M 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 Section 1.1) and the neurons were allowed to load with dye for 15 minutes at room temperature in a 100% 0 2 atmosphere. Changes in intracellular free Ca concentration were measured in experiments utilizing acutely dissociated adult rat hippocampal C A I neurons. To measure changes in [Ca 2 +] b the fluorescent indicator fura-2-AM (7 uM; 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 30 minutes. The prepared chamber (containing neurons loaded with BCECF or fura-2) was placed onto the stage of an inverted microscope. The neurons were then superfused continuously with either HEPES-buffered saline (Table 1, solution 1) or H C 0 3 7 C 0 2 - buffered saline (Table 2, solution 6), pH 7.35 at 37°C, at a rate of 2.0 ml min"1 for 15 minutes prior to the start of each experiment to ensure full de-esterification of the dye. The temperature-controlled perfusion chamber allowed for uninterrupted switching of perfusion solutions, e.g. normoxic to anoxic medium. When perfusing with H C 0 3 7 C 0 2 - buffered media, the atmosphere in the perfusion chamber contained 5% C 0 2 in balance air (normoxia) or 5% C 0 2 in balance Ar or N 2 (anoxia). 30 3.2 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 fluorescence intensities at alternating wavelengths of excitation at 488 nm and 452 nm (i.e. Im/I4S2)- The intensity of the 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 fluorophore, the excitation wavelengths employed were 334 nm and 380 nm, both of 2+ • which are sensitive to changes in [Ca ] i 5 although I3U increases with an increase in [Ca2+]j and I3S0 decreases with an elevation in [Ca2+]. In either case, because the intensities of fluorescence emissions 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; Bright et al, 1989; Silver et al, 1992). Throughout an experiment in which pH ( was being measured, the viability of the neuron was assessed by monitoring the stability of the fluorescence emission intensity during excitation at 452 nm (I452; pH-insensitive). Bevensee et al (1995) monitored rates of BCECF loss and morphological deterioration of acutely isolated post-natal rat hippocampal CA1 neurons and determined that the rate of change of 7452 is 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 31 (Bevensee et al, 1995). Consequently, neurons in which the rate of change of I452 was greater than 5% min'1 were excluded from analysis. The experimental apparatus consisted of a Zeiss Axiovert 10 microscope (Carl Zeiss Canada Ltd., Don Mills, ON) in conjunction with an Attofluor Digital Fluorescence 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 for BCECF, and was then reflected by a dichroic mirror (FT > 495 nm) into the objective (Zeiss L D Achroplan, n.a. 0.60, 40x) to excite the BCECF loaded into the neurons. Fluorescence emitted by the neurons was collected at 520 nm and measured by an intensified charge-coupled device camera. The camera gains at each excitation wavelength were set to maximize the image intensity, minimize the possibility of camera saturation, and were 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 minimize 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 the incident light at each excitation wavelength. In addition, a computer actuated high speed shutter restricted the exposure of the neurons to the U V light to periods when emitted fluorescence intensities at each excitation wavelength were being measured (usually once every 2 to 15 seconds). Finally, when using BCECF, a variable intensity 32 lamp control (Attoarc, Carl Zeiss Canada Ltd.) was employed to reduce the intensity of the 100 W mercury arc lamp by 50%. 3.3 Calculation of pH, and [Ca2+]{ 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., Redmond, WA), the respective background fluorescence intensities were subtracted from the raw emission intensities at each excitation wavelength from each ROI throughout the experiment to yield background-corrected ratios of emission intensities (BI4SS/BI452 in the case of BCECF; BI33JBI3S0 in the case of fura-2). Calibration of fura-2 signals was not attempted (see Church et al, 1998) and changes in [Ca2+]j evoked by experimental maneuvers are presented as changes in background-corrected I33JI3m ratio values. At the end of each experiment in which BCECF was employed, a one-point calibration was performed where the neurons were exposed to an high-K+ HEPES- buffered solution, pH 7.00 at 37°C (Table 1, solution 5) that contained 10 u M nigericin, a K 7 H + ionophore (Thomas et al, 1979). Nigericin, a charged electron carrier, balances cytoplasmic and extracellular [K+] and, in doing so, equilibrates pH 0 to pHj (Chaillet and Boron, 1985). Since external pH was titrated to pH 7.00, pHj was eventually clamped at pH 7.00 after 5-10 minutes of exposure to the calibrating solution. It has been suggested that the high KVnigericin technique may introduce errors when employed to measure absolute pH; (Boyarsky et al, 1996a and 1996b). In our hands, however, correction 33 factors provided by these authors failed to significantly affect steady-state pHj values estimated without correction. Furthermore, it is important to note that relative pHj changes evoked by an experimental maneuver are not affected by the application of the aforementioned correction factors (see Nett and Deitmer, 1996). For each experiment, the background-corrected ratio of emission intensities during the calibration period represented the BI4SS/BI452 ratio corresponding to pH 7.00. The experimentally-derived background-corrected ratios (BI4SI/BI452) were then divided by the ratio corresponding to pH 7.00, obtaining normalized ratios which were then converted to corresponding values of pHj using Equation 4, which is based on the Henderson-Hasselbalch expression for the dissociation of a weak acid pHj = log[(R n-R n ( m i n ))/(R n ( m a x )-R n)] + pK a (Equation 4) where R„ denotes the background-corrected ratio normalized to pH 7.00, Rn ( m a x ) and R^,,) represent the maximum and minimum obtainable values for the normalized ratio, and pK a is the -log of the dissociation constant of BCECF (for the derivation of Eq. 4 see Baxter, 1995). R n ( m a x ) , Rn(min) and pK a were derived from in situ calibration experiments (Fig. 2). In such experiments, cultured post-natal hippocampal neurons were exposed to a series of high-K+ HEPES-buffered solutions, each containing 10 u M nigericin, titrated to pH values ranging from pH 5.5 to pH 8.5 in 0.5 pH increments using 10 K O H or 1 M HCI. The average background-corrected ratios (BI4iS/BI452) for all neurons at each pH were normalized to the average ratio of background-corrected emission intensities at pH 7.00 34 and a pH titration curve was produced (Fig. 2B). The upper and lower asymptotes of this curve represent the R„ ( m a x ) and Rn (min), respectively, while the cytosolic pH on the abscissa corresponding to the point of inflection of the curve represents the measured pK a of BCECF (see Boyarsky et al, 1988; Baxter and Church, 1996). 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 dependent upon the temperature of the perfusate or 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 13 full calibration experiments used in analyzing all experiments, the values for Rn(ma!<), R„(mi„) and pK a (mean ± S.E.M.) were 1.89 ± 0.04, 0.43 ± 0.02 and 7.19 ± 0.02, respectively. 4 Experimental Procedures 4.1 Induction of anoxia In most cases, anoxia was induced by the addition of 2 m M sodium dithionite, Na 2 S 2 0 4 , an oxygen scavenger, to the superfusing medium (see Friedman and Haddad, 1993). Anoxic media was prepared immediately prior to use and the pH was adjusted as necessary. The partial pressure of oxygen was measured with a Radiometer A B L 500 blood gas analyzer calibrated for low Po2. In a solution containing 2 m M Na 2 S 2 0 4 , the Po2 was < 1 mm Hg (n=4). In some experiments, anoxia was induced by equilibrating solutions for 24 hours with 100% high purity Ar in HC03"-free, HEPES-buffered medium or with 5% C0 2 -95% high purity Ar in HC037C02-buffered medium. In this case, Po2 35 was also reduced to < 1 m M Hg. Given that the pH, response was not different when anoxia was induced either by the addition of 2 m M Na 2 S 2 0 4 (Fig. 7A) or the equilibration of the perfusing media with Ar for 24 hours (see Results, Section 4, Fig. 11), these data confirm that the pHj response to anoxia occurs in response to a reduction in Po2 and is not secondary to an additional property of 2 m M Na 2 S 2 0 4 . In order to induce hypoxia, a HC0 37C0 2-free, HEPES-buffered medium was equilibrated with 100% high purity Argon gas for one hour. The Po2 measured was 25 ± 1 mm Hg (n = 4). To induce a similar insult, a HC0 37C0 2-free, HEPES-buffered medium containing 0.5 m M N a ^ C ^ reduced the Po2 to 30 ± 3 mm Hg (n - 6). 4.2 Changes in steady-state pHx and rates of recovery ofpHx The effects of anoxia, alone or in combination with a pharmacological treatment or extracellular ion manipulation, on steady-state pHj were examined. In a H C 0 3 7 C 0 2 buffered medium, all known pH; regulating mechanisms present in hippocampal neurons are active, i.e. the Na+-dependent and Na+-independent HC0 3 /C1" exchangers and the Na7H + exchanger. In a nominally HC0 3-free, HEPES buffered medium, both HC0 3 7CT exchangers are inactive and Na7H + exchange is the dominant pHj regulating mechanism (Raley-Susman et al, 1991; Schweining and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998). By comparing results obtained in the presence or absence of HC0 3 " , the relative roles of HC03"-dependent and HC03'-independent pH; regulating mechanisms in mediating any change in steady-state pHj evoked by an experimental maneuver could be assessed. 36 At resting (physiological) pH i ? pHj regulating mechanisms present may or may not be active. For example, in cultured fetal rat hippocampal neurons, it has been shown that the Na+-independent HC037C1" exchanger is operational as an acid extrusion mechanism only at very low levels of pHj (pHj < 6.4) and not at "normal" resting pHj values (Baxter and Church, 1996). Furthermore, some agents (e.g. mitogens) can affect the activities of both acid-extruding and alkali-extruding pH; regulatory mechanisms to a similar extent, leaving steady-state pH; unchanged (e.g. Ganz et al, 1989). Therefore, the effects of anoxia were also assessed on rates of recovery of pHj after imposed intracellular acid loads. Intracellular acidifications were induced by the N H 4 + prepulse technique (Boron and De Weer, 1976). This involves exposing the neurons to a medium containing 40 m M NH 4C1 (Table 1, solution 4), a maneuver which causes an intracellular alkalinization. Upon washout of the NH4+-containing medium, pH; undershoots its initial steady-state value. From this acidified level of pH i 5 the neuron utilizes acid extrusion mechanisms to recover to the initial resting level of pHj (Roos and Boron, 1981; Boron, 1989). In each experiment in which rates of pH; recovery from imposed internal acid loads were assessed, a given neuron was subjected to two intracellular acid loads. The initial acid load was used to calculate the control rates of pHj recovery for a given neuron while the second acid load, and subsequent recovery of pH s , was performed either during or following a transient anoxic period. The control rates of pH s recovery were compared to the rates of pHj recovery under a given test condition at the same corresponding absolute values of pH s . The rationale for such a comparison is that the activities of acid 37 extrusion mechanisms are dependent upon the absolute pHj (see Boyarsky et al, 1988). Since the activities of acid extrusion mechanisms are 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 activities of acid extrusion mechanisms (and, thus, rates of recovery of pH;) are accounted for. 5 Analysis of Data 5.1 Analysis of steady-state changes in pH, In order to compare the steady-state pHj response to anoxia under varying conditions, a variety of parameters were measured (Fig. 3). The time required for the initiation of the acidic shift (A) is the time between the beginning of the anoxic period and the point at which pHi begins to decrease. The time required for the maximum acidic shift (B) is the time between the beginning of the anoxic period and the point at which the lowest pHj value occurred. The time required for the rise of pHj during the anoxic period (C) is the time between the beginning of the anoxic period and the point at which pHj begins to increase in the continued absence of 0 2 . The time required for the alkaline shift (D) is the time between the end of the anoxic period and the point at which the highest pHj value first occurred. The magnitude of the acidic shift (E) is the difference between the pre-anoxic steady-state pHj value and the lowest pHj value observed during the anoxic period. The magnitude of the recovery during anoxia (F) is the difference between the pHj value observed immediately prior to the return to normoxic medium and the lowest pHi value observed during the anoxic period. This measurement was employed to 38 calculate the percentage recovery of pH; towards pre-anoxic resting levels in the absence of 0 2 according to the equation: % pi t recovery = 100*(F/E). The magnitude of the alkaline shift (G) is the difference between the highest pH; value observed after the anoxic period and the pre-anoxic pHj value. In order to inhibit the activity of the Na7H + exchanger, reduced-[Na+]0 media were employed, in which all but 2 m M extracellular Na + was iso-osmotically replaced with the membrane-impermeant cation, TY-methyl-D-glucamine. In acutely dissociated hippocampal neurons, this maneuver produces a rapid intracellular acidification followed by a slow increase in pH; towards resting pHj values (Fig. 4A; see Bevensee et al, 1996). In order to allow an accurate assessment of the pHj response to anoxia under reduced-Na+ conditions, this slow pH; recovery was fit, utilizing the least squares method (TableCurve 2D, Jandel Scientific, San Rafael, CA), to a double exponential curve having the equation, y = a + b( 1 -<cx))+d( 1 -eO) (Equation 5) where b and c are exponential parameters of the first exponential portion, and d and e are exponential parameters of the second exponential portion. A l l data points from the point of maximum acidification in the reduced-Na+ solution until the induction of anoxia were fit with this equation. The curve was extrapolated through the time points over which anoxia was superimposed, giving a baseline from which the necessary parameters (identified above) could be measured (Fig. 4A). The double exponential curve was 39 deemed a sufficient fit with r2 values of > 0.80 and a composite residuals graph showed points that are uniformly distributed around the zero line (Fig. 4B). Experimentally-evoked changes in any of the steady-state pH; parameters measured in response to anoxia were assessed with unpaired, two-tailed Student's t tests. Errors are expressed as S.E.M and n values refer to the number of neurons from which data were obtained. Any net difference was deemed to be significant i f the resultant p value was <0.05. 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 pH; vs. time records, a moving average (period = 5) was applied to all traces shown (see Boyarsky et al, 1988; Baxter and Church, 1996). 5.2 Analysis of the rates of recovery ofpH^from imposed intracellular acid loads In analyzing the recovery of pH; from an imposed intracellular acid load, the recovery portion of the experiment was fitted, utilizing the least squares method (SigmaPlot v. 3.02, Jandel Scientific), to a single exponential function of time having the format p H ^ a + bO-O, (Equation 6) 40 where a, b, and c are the exponential parameters. The differentiated form of Equation 6 yields the rate of change of pH; dpH/dr = bc(ect) (Equation 7) from which the instantaneous rates of pHj recovery (dpH/dr) were calculated at 0.05 unit intervals of pH; from the point of maximum acidification following an NH 4 + prepulse until recovery was -80% complete (see Baxter and Church, 1996; Smith et al, 1998). For each neuron, a paired experiment, consisting of two consecutive intracellular acid loads, was performed (see Section 4.2). At each corresponding value of pH i5 the percentage difference between the control rate of pHi recovery and the rate of pHj recovery either during or following a transient period of anoxia was determined. An average of the resultant percentage differences, over the range of pHj values, was calculated and utilized for description of the data. To determine the intrinsic percentage differences in rates of pHj recovery between two consecutive acid loads, paired experiments were performed under control conditions, i.e. in the absence of any experimental treatment (Fig. 5A). The instantaneous rates of pHj recovery following the first acid load were grouped separately from the instantaneous rates of pHs recovery following the second acid load. The percentage differences between the average instantaneous rates of pHj recovery from the first and second acid loads at corresponding pHj values was calculated and the mean percentage difference over the range of pH; values was obtained. Based on data from 18 neurons, the average difference 41 observed in the rates of pH r recovery from the second acid load compared to the first acid load was a -1.20 ± 21.3% increase (mean ± S.D.; Fig. 5B). Therefore, rates of pHj recovery obtained either during or following an anoxic insult were considered different from the control rates of pHi recovery only if they displayed either an average increase or decrease of 43% (i.e. ~ 2 S.D.) or more as compared to the control rates of pH ; recovery. Only neurons which exceeded these criteria underwent further analysis. The instantaneous rates of pH; recovery under control conditions were grouped separately from the instantaneous rates of pR, recovery under the influence of a test condition and a formal statistical comparison between these two groups was performed using an unpaired, two-tailed Student's /-test. If the resultant p value was < 0.05, then the rates of pHi recovery from an imposed intracellular acid load were considered to have been altered significantly in response to the test maneuver. In summary, data obtained from each neuron were initially assessed on the basis of the average percentage difference between "control" and "test" rates of pHj recovery to determine i f the criteria established for inclusion were satisfied. Control and test data from each accepted neuron were then grouped separately and compared at absolute pHj values. If the Student's Mest returned a p value < 0.05 at a given pH; value, then the changes in the rates of.pHj recovery are most likely due to an alteration in the rate of acid extrusion. 42 Table 1: Composition of HEPES-buffered experimental solutions (all concentrations in mM) Solution 1 Standard 2 Low Na + 3 Ca 2 + free 4 NH4C1 5 High K + NaCl 136.5 2.0 136.5 96.5 - KC1 3.0 3.0 3.0 3.0 - CaCl 2 2.0 2.0 - 2.0 2.0 NaH2PO„ 1.5 - 1.5 1.5 1.5 MgS0 4 1.5 1.5 3.0 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 + - 136.0 - - - NH4C1 - - - 40.0 - HEPES 10.0 10.0 10.0 10.0 10.0 Titrated with: 10M NaOH 1.0 M HC1 10M NaOH 10 M NaOH 10 M KOH Ca +-free medium also contained 200 uM ethylene glycol-bis((3-aminoethyl ether) N, N, TV, 7Y- tetraacetic acid (EGTA). Abbreviations: Na Glu, sodium gluconate; K Glu, potassium gluconate; NMDG + , W-methyl-D- glucamine 43 Table 2: Composition of HC037C02-buffered experimental solutions (all concentrations in mM) Solution 6 7 Standard Standard pH 7.35 at 37°C pH7.35at22°C NaCl 127.0 117.5 NaHC0 3 19.5 29.0 KC1 3.0 3.0 CaCl 2 2.0 2.0 NaH 2P0 4 1.5 1.5 MgS0 4 1.5 1.5 D-glucose 17.5 17.5 Al l HC03"-containing solutions were equilibrated with 5% C0 2 in balance air (normoxia) or balance argon or nitrogen (anoxia). i 44 Table 3: List of pharmacological agents employed Stock Concentration Solvent Test Concentration Storage Bafilomycin A] 2 mM DMSO 1 - 2 u M -20 °C 4-o> 5-hydroxycinnamate 500 mM 4 DMSO: 5 - 10 pM N/A 1 methanol (v:v) DIDS 100 mM DMSO 200 uM N/A Isoproterenol 50 mM ultra-pure water 10 pM -60 °C Nigericin 10 mM ethanol 10 pM -60 °C Noradrenaline 50 mM ultra-pure water 10 pM -60 °C Omeprazole 25 mM DMSO 50 pM N/A Rp-cAMPS 50 mM ultra-pure water 50 pM -20 °C Tetrodotoxin 100 pM ultra-pure water 0.5 - 1 pM -2 °C All agents were supplied by Sigma Chemical Company, with the exception of Bafilomycin A] (a generous gift from Dr. V . Palaty, Department of Anatomy, UBC) and Rp-cAMPS (Biolog Life Science Institute, La Jolla, CA). Abbreviations: DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid; DMSO, dimethylsulphoxide. 45 Figure 1. Acutely dissociated adult rat hippocampal CA1 neurons Neurons were isolated according to procedures described in Section 1.1 and were chosen for study based on morphological criteria established by Schwiening and Boron (1994), i.e. a smooth, non-granular appearance; a single major process (presumably an apical dendrite) projecting from one pole of the soma which was at least three times the length of the diameter of the cell body; and the presence of two or more smaller processes (basal dendrites) at the opposite pole. 46 47 Figure 2. Sample calibration plot for B C E C F A . Cells were exposed to HC0 37C0 2-free, HEPES-buffered solutions containing 10 u M nigericin at pH 0 (and therefore pH;) 5.54, 6.04, 6.51, 7.00, 7.51, 7.98, and 8.48. The duration of each exposure is indicated by the bars above the trace, which is the mean of data obtained from 17 cultured post-natal rat neurons recorded on a single coverslip. B . Plot of pH, against the resulting normalized ratio (R„). Standard error bars are indicated («=3 coverslips). Where absent, error bars lie within the symbol area. The curve is a result of a non-linear least squares regression fit to Equation 4. For this particular calibration, the values of R„ ( m a x ), R„ ( m i n ), and pK a were 2.0115, 0.4346, 7.2526, respectively.  49 Figure 3. Experimental parameters measured to assess the steady-state p l i response to a transient period of anoxia The pHi response of an acutely dissociated adult rat hippocampal C A I neuron to a 5 min period of anoxia is illustrated. The time required for the initiation of the acidic shift (A) is the time between the beginning of the anoxic period and the point at which pIL began to decrease. The time required for the maximum acidic shift (B) is the time between the beginning of the anoxic period and the point at which the lowest pHj value occurred. The time required for the initiation of the rise of pHj (C) during the anoxic period is the time between the beginning of the anoxic period and the point at which pH, began to increase in the continued absence of 0 2 . The time required for the alkaline shift (D) is the time between the end of the anoxic period and the point at which the highest pH ; value first occurred. The magnitude of the acidic shift (E) is the difference between the pre-anoxic steady-state pH ; value and the lowest pHj value observed in the anoxic period. The magnitude of the recovery during anoxia (F) is the difference between the pH ; value observed immediately prior to the return to normoxic medium and the lowest pH, value observed during the anoxic period. The magnitude of the alkaline shift (G) is the difference between the highest pH; value observed after the anoxic period and the pre- anoxic steady-state pH; value.  51 Figure 4. The pHj response upon exposure to a reduced-[Na + ] 0 medium A . Upon exposure to a 2 m M [Na+]0, NMDG+-substituted, HEPES-buffered medium, the initial, steady-state pHj acidified by ~ 0.50 pH units and gradually recovered. pH, reached a plateau value of ~ 7.40 after ~ 20 min during the continued perfusion with a reduced- Na + medium. A l l data points from the point of the maximum acidification were curve fit to a double exponential curve (solid line). The raw data points are overlying the extrapolated curve (1^=0.99). B. Plotted are the average residual values (the magnitude of the difference between a raw data point and the curve fitted data point) against time from the point of peak acidification upon exposure to 2 m M [Na+]0, NMDG+-substituted, HEPES-buffered media until the end of the curve-fitted data points. A. 7.5 2 m M [Na + ] 0 , NMDG+-substituted point of peak acidification 53 Figure 5. Control acid load recoveries during perfusion with HC0 3 7C0 2 - f ree , HEPES-buffered medium A . In order to determine whether anoxia influences the rates of pHj recovery from an imposed intracellular acid load as compared to the control rates of pH ; recovery, the intrinsic differences between the rates of pH ( recoveries of two consecutive, control acid loads must be accounted for. In the example shown, an initial acid load was performed and, after pHj had recovered to a new steady-state value, a second acid load was conducted. The percentage differences between the rates of pHt recovery following the initial acid load and the rates of pH; recovery following the second acid load at each corresponding absolute value of pH; were calculated. An average of the percentage differences at each pH ; value was then determined. In this example shown, the rates of pH, recovery from the second acid load were on average 27% faster than the rates of pH ( recovery from the initial acid load. B . The graph represents a comparison between the rates of pH; recovery from an initial (O) and from a second (•) acid load, both obtained under control conditions, at the same absolute values of pH; («=18). The rates of pH ( recovery were evaluated at 0.05 pH unit intervals of pH ( and error bars represent S.E.M. Both sets of data were independently fitted with a linear least-squares regression line. The average percent difference, calculated over the range of pHj values shown, was -1.20 ±21 .3% (mean ± S.D.). Time (min) 0.004 0.002 0.000 55 R E S U L T S Using acutely dissociated adult rat hippocampal CA1 neurons, I initially characterized the changes in steady-state pHj which occur in response to a transient period of anoxia. Next, the underlying mechanisms responsible for the production of the anoxia-evoked changes in pHj were examined, with particular emphasis on the role of changes in the activity of the N a + / H + exchanger. Finally, the effects of such variables as ambient temperature, extracellular pH, extracellular glucose concentration and the activity of the c A M P / P K A second messenger system on the pHj response to anoxia were examined. Each of these variables have previously been shown to affect neuronal viability following a transient anoxic insult. 1 Distribution of steady-state intracellular pH under normoxic conditions In HC0 37C0 2-free, HEPES-buffered media at pH 7.35, the mean steady-state pH; value was 7.30 ± 0.21 (range pHj 6.40 - 7.80; n=694; Fig. 6A). The distribution was best fitted by a bimodal Gaussian distribution with means at pH, 6.93 ±0.17 and 7.37 ± 0.14. In HC0 37C0 2-buffered media, pHj was distributed, in a Gaussian manner, around a mean of 7.30 ±0.17 (range pH ; 6.90 - 7.60; «=83; Fig. 6B). The mean steady-state pH ; values, in the absence and presence of HC0 3 " , were not statistically different, suggesting that the predominant mechanism involved in the maintenance of steady-state pH; in acutely dissociated adult rat hippocampal CA1 neurons at 37°C is HG03"-independent and likely to be the N a + / H + exchanger, as previously described (Baxter and Church, 1996; Bevensee etal, 1996; Smith et al, 1998). 56 2 Characterization of the intracellular pHresponse to anoxia 2.1 Steady-state changes in intracellular pH evoked by anoxia under HC03'/C02-free, HEPES-buffered conditions The pHj changes evoked by a 5 min period of anoxia were characterized by three phases (Fig. 7; Table 4). In a HC0 37C0 2-free, HEPES-buffered medium (pH 7.35), a 5 min period of anoxia induced an initial acidic shift in pHj of 0.16 ± 0.01 pH units («=28), with the peak acidification occurring 2.5 ± 0.2 min following the start of anoxia. In the continued absence of oxygen, pHj recovered by 0.05 ± 0.01 pH units, a 40 ± 5% recovery. Upon the return to normoxia, there was a further internal alkaline shift in pHj of 0.20 ± 0.02 pH units above the initial pre-anoxic steady-state pHj value which reached a maximum at 9.5 ± 1.1 min after the end of anoxia. The magnitude of the internal acidification observed during a 5 min period of anoxia showed a positive correlation to the initial pre-anoxic steady-state pHj of the cell (r2 = 0.70). No similar correlation was observed between the pre-anoxic steady-state pH ; value and the magnitude of the post- anoxic alkalinization, nor was there any apparent correlation between the magnitude of the anoxic acidification and the magnitude of the post-anoxic alkalinization (r2 = 0.25 and 0.20, respectively). A change in the duration of the anoxic period from 5 min to 3 or 10 min did not influence significantly the magnitude of the intracellular acidification observed during anoxia (Fig. 8; Table 4). However, the magnitudes of the rise in pH ; observed during anoxia and the post-anoxic alkalinization increased with an increasing duration of anoxia, 57 being larger during/following a 10 min period than compared to during/following a 3 min period of anoxia (Table 4; p < 0.05 in each case). 2.2 Steady state changes in intracellular pH evoked by anoxia under HC03'/C02-buffered conditions Transient anoxia of 3, 5, or 10 min duration induced a qualitatively similar triphasic partem of pHj changes under HC0 37C0 2-buffered conditions as was observed under HC0 37C0 2-free conditions. A 5 min period of anoxia induced an intracellular acidification that partially recovered in the continued absence of oxygen and, upon the return to normoxia, there was an intracellular alkalinization above pre-anoxic steady-state pH, values (Fig. 9B; Table 5). Under HC0 37C0 2-buffered conditions, the magnitudes of the anoxic acidification and the post-anoxic alkalinization were not correlated with either the initial resting pHj observed prior to anoxia or with each other (r2 = 0.61, 0.29, 0.35, respectively). In a manner similar to that observed under HEPES-buffered conditions (see Section 2.1), the magnitude of the per-anoxic rise in pH s increased as the duration of the anoxic period increased from 3 to 5 to 10 min (Table 5). In response to a 5 min period of anoxia, none of the parameters measured were influenced by the presence or absence of H C 0 3 ' . In response to a 3 min period of anoxia, although the magnitude of the intracellular acidification seen during the anoxic period was smaller in the presence of H C 0 3 " (Table 5; /?<0.05), all other aspects of the pH s response were similar in the presence and absence of HC0 3 " . However, following a 10 min period of anoxia, the magnitude of the post-anoxic alkalinization was statistically 58 larger under HC0 37C0 2-free, HEPES-buffered conditions compared to that observed under HC0 37C0 2-buffered conditions (p<0.05). In HC0 3/C0 2-buffered media, the addition of 200 u M DIDS failed to affect this difference (the rise in pHj observed following a 10 min period of anoxia in the presence of DIDS was 0.23 ± 0.03 pH units (n=\2) vs. 0.19 ± 0.02 pH units for the magnitude of the post-anoxic alkalinization in the absence of DIDS; p>0.\). A l l other aspects of the pHj response to a 10 min period of anoxia (with the exception of the time required to reach the maximum post-anoxic internal alkalinization; Table 5) were not influenced by the presence or absence of extracellular HC0 3 " . Therefore, potential changes in the activities of HC03"-dependent pHj regulating mechanisms appear to contribute little, i f at all, to the pHj response to anoxia. Consequently, all subsequent experiments were performed under H C 0 3 7 C 0 2 - free, HEPES-buffered conditions. BI452 remained stable during and following transient anoxic periods, indicating that the pHj shifts observed were not an artifact caused by changes in BI452 consequent upon a deterioration in membrane integrity (Fig. 7B; see Bevensee et al, 1995; Smith et al, 1998). However, the morphology of the cells did show visible changes during the course of an experiment. In agreement with previous reports (Florine-Castel et al, 1991; Friedman and Haddad, 1994a), anoxia induced the formation of membrane blebs on the surface of the soma, and along the length of the apical and basal dendrites. In more severe cases, the membrane blebs either fused together, causing the cell body to swell, or the blebs would detach from the plasma membrane. Under phase-contrast microscopy, following a transient period of anoxia, the cytosol of the cell appeared granular. 59 Although the present study did not evaluate the mechanisms initiating the observed anoxia-induced neuronal damage, possibilities include an increase in [Na+] i 5 a stimulation of cytoskeletal proteases, phospholipases, and/or a change in plasma membrane structure (Herman et al, 1990; Arai et al, 1991; Florine-Casteel et al, 1991; Friedman and Haddad, 1994b). 3 Characterization of the intracellular calcium response to anoxia Intracellular calcium increased in response to a transient period of anoxia. Experiments were performed in HC0 37C0 2-free, HEPES-buffered media at pH„ 7.35. The average resting BI334/BInQ value prior to the induction of anoxia was 0.7 ±0.15 (Fig. 10; n=3). This value increased sharply beginning 1.6 ± 0.4 min after the induction of anoxia and reached a maximum value of 2.74 ± 0.43 at 3.1 ± 0.5 min following the induction of anoxia. After the return to normoxia, the ratio remained elevated for up to 15 min (the maximum period recorded). A 3 min period of anoxia induced a similar 2+ [Ca ]j response, the time course and magnitude of which were statistically not different to the respective values measured during and after a 5 min period of anoxia (n=3; data not shown). 4 Validation of the use ofNa^SJD^ to induce anoxia In order to control for any properties of sodium dithionite unrelated to its ability to reduce the Po 2 of a given media, the effects on pHj of exposing neurons to physiological media equilibrated with high-purity Argon gas were investigated. 60 Initially, a HC0 37C0 2-free, HEPES-buffered medium was bubbled vigorously with 100% high purity Argon gas for one hour, a maneuver which reduced Po 2 to 25 ± 1 mm Hg (n = 4). This procedure is often employed to induce "anoxia" (e.g. Krnjevic and Walz, 1990; Donnelly et al, 1992; Pirttila and Kauppinen, 1992; O'Reilly et al, 1995); however, the present data suggest that exposure to this solution induces an hypoxic insult (see Arai et al, 1991; Fujiwara et al, 1992). Out of 14 cells examined, 6 cells responded to a 5 min period of hypoxia with an acidification during the hypoxic period of 0.07 ± 0.03 pH units followed by an alkalinization of 0.25 ±0 .11 pH units above pre-hypoxic - resting levels upon the return to normoxia. The remaining 8 cells did not show any change in pHj in response to perfusion with the hypoxic medium. A 0.5 m M solution of N a 2 S 2 0 4 in a HEPES-buffered medium, in the absence of equilibration with Argon, reduced the Po 2 in the medium to 30 ± 3 mm Hg (n = 6), a level not statistically different from that reached after equilibration with 100% high purity Argon gas for one hour. The pH, response to a 5 min period of hypoxia induced with the 0.5 m M Na^^-containing medium was not statistically different in any respect from that induced by perfusion with Na2S204-free medium equilibrated with Argon for one hour (0.03 ± 0.01 pH units and 0.29 ± 0.04 pH units for the magnitudes of the acidification and alkalinization, respectively, evoked by a 5 min period of anoxia induced with 0.5 m M Na 2 S 2 0 4 ; n=6). When the Po 2 was reduced to < 1 m M Hg by equilibration of a H C 0 3 7 C 0 2 - buffered solution with 95% Argon/5% C 0 2 for > 18 h, a 5 min period of anoxia produced a pHj response which was identical to that observed when the Po 2 was reduced to < 1 mm Hg by the addition of 2 m M Na 2 S 2 0 4 (compare Fig. 9B and Fig. 11; 0.12 + 0.01 pH units 61 vs. 0.18 ± 0.02 pH units, respectively, for the acidification seen during the anoxic period, and 0.18 ± 0.07 pH units vs. 0.16 ± 0.05 pH units, respectively, for the post-anoxic alkalinization; n>4 in each case). Thus, the pHj response to anoxia is not an artifact associated with the use of Na 2 S 2 0 4 to induce anoxia but rather, is determined by the reduction in the Po2 in the experimental media. This latter conclusion is reflected by the fact that both the acidification observed during anoxia and the post-anoxic alkalinization were significantly different when the hypoxic (Po2 = 25-30 mm Hg) and the anoxic (Po2 = 0 mm Hg) responses were compared. While the per-anoxic acidification was smaller, the post-anoxic alkalinization was larger under hypoxic conditions compared to anoxic conditions (0.04 ± 0.01 pH units vs. 0.17 ± 0.01 pH units under hypoxic and anoxic conditions, respectively, for the per-anoxic acidification; 0.28 ± 0.04 pH units vs. 0.16 ± 0.03 pH units under hypoxic and anoxic conditions, respectively, for the post-anoxic alkalinization; p<0.05 in each case). A second concern regarding the method of inducing anoxia with Na2S204 was that the addition of Na^CX, may alter the pH of the anoxic test media, even after retitration. Arguing against this possibility, however, is that the pH of the anoxic media measured at the end of an experiment decreased by 0.021 ± 0.003 pH units (n=24) from the pH value measured prior to its use, a reduction which cannot account for the magnitude of the cellular pHj shifts seen (see Friedman and Haddad, 1993; Diarra et al, 1998). Thus, the use of 2 m M Na 2 S 2 0 4 provides an effective anoxic insult and, therefore, the addition of 2 m M Na 2 S 2 0 4 was employed to induce anoxic insults in all subsequent experiments. 62 5 Mechanisms underlying the intracellular pH response to transient anoxia 2+ Having characterized the pHj and [Ca ]j responses of isolated C A I hippocampal neurons to transient anoxic periods of varying durations, the next series of experiments examined possible changes in the activities of pHj regulating mechanisms that may be involved in the production of the observed steady-state pH; response. 5.1 RoleofCa2+ 2+ + Given the close interrelationship between [Ca ]•, and [H ]j (Meech and Thomas, 1977; Ives and Daniel, 1987; Ou-Yang et al, 1994a and 1995), it was necessary to assess 2+ the possible role of changes in [Ca ]j to the changes in pHj evoked by anoxia. Initially, 2+ the effect of the removal of extracellular Ca on the pH ; response to anoxia was 2+ examined. Exposure to a HC0 3VCCyfree, HEPES-buffered, Ca -free solution containing 200 p M E G T A caused an average increase in steady-state pH; of 0.11 ± 0.05 pH units (Fig. 12A; «=6). Once a new steady-state pH; value was reached, a 5 min period 2+ of anoxia imposed under Ca -free conditions induced an intracellular acidification of 0.21 ± 0.04 pH units, reaching this maximum at 2.0 ± 0.4 min into the anoxic period (Fig. 12B). In the continued absence of oxygen, pHj recovered by 0.06 ± 0.01 pH units, a 40 ± 11% recovery. Upon the return to normoxia, there was an intracellular alkalinization of 0.18 ± 0.04 pH units above pre-anoxic resting pH ( levels, which reached a maximum value at 6.4 ±1 .3 min following the return to normoxia. Neither the time course nor the magnitude of any phases of the pHj response to anoxia were significantly different from 2+ those seen in the presence of Ca under identical buffering conditions (see Fig. 7A; 63 Table 4). Thus, the pH> response to a 5 min period of anoxia is independent of C a 2 + influx. This conclusion is supported by the fact that the internal acidification observed during anoxia began prior to the start of the anoxia-evoked rise in [Ca ]j (1.0 ±0 .1 min vs. 1.6 ± 0.4 min, respectively; p<0.05). The effect of the removal of extracellular C a 2 + on the [Ca2+]j response to a 5 min period of anoxia was also examined. The average resting BI3MIBI3m value prior to the 2+ removal of extracellular Ca was 0.56 ± 0.04 (n=6). Exposure to a HC0 3 ' /C0 2-free, 2+ HEPES-buffered, Ca -free solution caused a 0.30 ± 0.02 decrease in this value (Fig. 13). 2+ Under Ca -free conditions, a 5 min period of anoxia induced a 0.02 ± 0.01 increase in the ratio value, an increase which was significantly smaller than that observed in the 2+ presence of extracellular Ca under identical buffering conditions (see Fig. 10, Section 3; p<0.05). Interestingly, in 4/6 cells examined, there was a small post-anoxic increase in 2+ the 5/334/5/380 ratio value of 0.09 ± 0.02 which may reflect Ca release from intracellular stores, although this was not investigated. Thus, the [Ca2+]j response to transient anoxia is dependent on the presence of extracellular Ca 2 + ; however, the pH ; response to anoxia appears to be independent of any changes in [Ca 2 +]i. 5.2 Role of the / Y a V / T exchanger 5.2.1 Technique for isolation ofNa+/lT exchange activity The isoform of the N a + / H + exchanger present in hippocampal neurons is insensitive to amiloride, amiloride derivatives (e.g. dimethylamiloride, 5-(N-ethyl-N- isopropyl)amiloride), and benzoyl guanidinium compounds (e.g. HOE 694), agents which 64 inhibit N a + / H + exchange activity in peripheral cell types (Raley-Susman et al, 1991; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998). Thus, in order to assess the role of the N a + / H + exchanger in the production of the steady-state pHj response to anoxia, all but 2 m M extracellular N a + was iso-osmotically replaced with /V-methyl-D- glucamine (NMDG + ) . Upon exposure to this reduced-Na+ solution, pHj acidified but then gradually recovered to a value approximating the initial steady-state pHj observed in the presence of normal extracellular N a + (see Bevensee et al, 1996). In 7 cells, pHj acidified by 0.50 ± 0.04 pH units, followed by a 99.4 ± 0.5% recovery of pH, towards the initial pHj value after 30 ± 3 min in the presence of reduced-Na+ media (Fig. 14A). The influence of the residual 2 m M extracellular N a + on the alkalinization process was assessed. Under external Na+-free conditions (NMDG+-substitution), pHj initially fell by 0.45 ± 0.10 pH units and recovered to 102 ± 2% of the initial resting pHj value over 24 ± 6 min (n=5). There was no statistical difference between either the magnitude of the internal acidification nor the degree of alkalinization observed under the different conditions of [Na+]0, a finding which suggests that the Na7H + exchanger was inactive even in the presence of 2 m M external Na + . This finding is consistent with the previous report that the apparent K„, of the Na7H + exchanger in rat hippocampal neurons for extracellular N a + is 23-26 m M (Raley-Susman et al, 1991). Possible mechanisms mediating the rise in pH; under reduced or zero-[Na+]0 conditions are investigated in Section 11. 65 5.2.2 Effects of Na+/tf exchange inhibition on steady-state pH{ changes evoked by anoxia In the analysis of experiments that included a 5 min period of anoxia imposed under reduced-[Na+]0 conditions, the pH s recovery in the reduced-Na+, N M D G + - substituted medium was curve-fit with a double-exponential equation (see Materials and Methods, Section 5.1) and extrapolated through the anoxic period to estimate the baseline pHj from which the effects of the anoxic insult on pH, could be derived. A 5 min period of anoxia induced an intracellular acidification of 0.11 ± 0.01 pH units followed by a per- anoxic rise in pH, of 0.04 ± 0.01 pH units, a 34 ± 10% recovery (w=20; Fig. 14B; Table 6). Upon the return to normoxia, there was an intracellular alkalinization of 0.12 ± 0.02 pH units above the steady-state pH ; value observed prior to the induction of anoxia. The magnitudes of the per-anoxic acidification and the post-anoxic alkalinization, as well as the time required to reach the maximum post-anoxic alkalinization, were significantly reduced from values obtained in the presence of normal external N a + under identical buffering conditions (p<0.05 in each case). As a result of the prolonged duration of these experiments, the time over which recordings could be maintained in the post-anoxic period was often shorter than seen in the presence of normal extracellular Na + . Thus, to ensure that the reduction in the magnitude of the post-anoxic alkalinization was not secondary to a change in its time course, the magnitude of the post-anoxic alkalinization was measured at fixed time points following an anoxic period in the presence or absence of normal extracellular Na + , and this possibility was found not to be the case (data not shown). Taken together, the above findings indicate that the reduction of extracellular 66 Na + by substitution with N M D G + , a maneuver that inhibits the N a + / H + exchanger, attenuates the magnitudes of the per-anoxic intracellular acidification and the post-anoxic alkalinization. Unlike N-methyl-D-glucamine, extracellular L i + can act as a substrate for the N a + / H + exchanger (Kinsella and Aronson, 1981). Thus, experiments were performed with 2 m M Na + , Li+-substituted media. Exposure to this solution produced an intracellular acidification of 0.34 ± 0.02 pH units («=13) that, over a period of 11 ± 2 min, recovered to 100 ± 0.3% of the initial steady-state pHj value observed prior to the reduction in [Na+]0 (see Baxter and Church, 1996; Smith et al, 1998). After pIL reached a new steady-state plateau level, a 5 min period of anoxia induced an intracellular acidification which recovered slightly during the anoxic period and, following the return to normoxia, there was an intracellular alkalinization (Fig. 15; Table 6). Under these conditions, the magnitudes of both the per-anoxic acidification and the post-anoxic alkalinization were statistically larger than those seen when N M D G + was employed as the N a + substitute but were not significantly different from those values measured in the presence of a normal concentration of extracellular Na + . Together, these findings strongly support a role for the N a + / H + exchanger in the production of the pHj response to a 5 min period of anoxia. 5.2.3 Anoxia-evoked changes in the activity of the Na+/fT exchanger The differences observed between the effects of anoxia on steady-state pH s under N M D G + vs. Li+-substituted conditions suggest strongly that changes in the activity of the 67 Na7H + exchanger contribute to the steady-state pHj response to anoxia in acutely dissociated adult rat hippocampal CA1 neurons. Therefore, in the next series of experiments, I assessed directly whether there was a change in the activity of the N a + / H + exchanger either during or following a transient anoxic period. To do so, paired intracellular acid loads were performed in HC0 3 /C0 2-free, HEPES-buffered media, under which conditions the recovery of pHi is primarily mediated by the Na7H + exchanger (Raley-Susman et al, 1991; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998). Following an initial, control acid load, a second acid load was performed either during or following a 5 min period of anoxia, at a time when the steady- state pHj was decreased or increased, respectively. In the first set of experiments, the second acid load was performed during the anoxic period. In 8 cells, there was an overall 44 + 3% decrease in the average rate of pHj recovery from intracellular acid loads imposed during anoxia compared to the pre-anoxic control rates of pH, recovery established in the same cells (Fig. 16). The rates of pHj recovery were plotted against the absolute pH ( at which they were measured. At the same absolute pH, values, the rates of recovery of pHj were significantly slower during the anoxic period compared to the corresponding rates under normoxic conditions (Fig. 17; j»<0.05 at all pH, values except at pH ; 6.95). The slope of the linear regression line relating rates of pHj recovery to absolute pH i ? an indication of transporter activity, was 3 1 3 1 reduced from -21.88 x 10" s" under normoxic conditions to -13.90 x 10" s" under anoxic conditions, a 36% reduction in slope. There was also an acidic shift in the pHj dependence of pH, recovery, with the x-intercept of the regression line shifting from pHj 68 = 7.50 under normoxic conditions to pHj = 7.43 under anoxic conditions. These results support the possibility that, during a transient anoxic period, there is both a decrease in the rate of activity of the Na7H + exchanger and an acidic shift in the pH; sensitivity of the antiport. In the next set of experiments, the second acid load was performed following a 5 min period of anoxia. In 17 cells, there was a 111 ± 18% increase in the overall rate of pH, recovery from intracellular acid loads imposed following a 5 min period of anoxia (Fig. 18). Rates of pH s recovery were plotted against the pHj value's at which they were measured, and linear regression lines were obtained relating the rates of pHj recovery to absolute values of pH; under pre-anoxic and post-anoxic conditions. At every absolute value of pHj, the rate of recovery of pHj was significantly faster following a 5 min period anoxia when compared to the corresponding pre-anoxic control rate of recovery established in the same cells (Fig. 19; p<0.05 at all pHi values). The slope of the linear 3 1 regression line was increased from -17.74 x 10' s' under pre-anoxic control conditions to 3 1 -21.43 x 10"J s" following a 5 min period of anoxia, a 121% increase in slope, indicating an increase in the activity of the N a + / H + exchanger. In addition, there was a shift in the x- intercept of the regression line from pH; = 7.45 under pre-anoxic conditions to pHj = 7.60 under post-anoxic conditions, indicating an alkaline shift in the pHj sensitivity of the exchanger. Thus, following a transient anoxic period, there is both an increase in rate of the activity, and an alkaline shift in the pH, sensitivity, of the NaTFf exchanger. 69 5.3 The effects of tetrodotoxin (TTX) During anoxia, there is a dramatic increase in intracellular N a + which may reduce the activity of the Na7H + exchanger (Friedman and Haddad, 1994b). As N a + influx via TTX-sensitive, voltage-activated N a + channels has been reported to mediate, at least in part, this rise in [Na+]i (Haddad and Jiang, 1993; Balestrino, 1995; Gleitz et al, 1996; Fung and Haddad, 1997), the influence of 0.5 - 1 u M TTX on the steady-state pH s response to anoxia and the rates of pH s recovery following intracellular acid loads imposed during an anoxic period were examined. Experiments were performed in H C 0 3 " /C02-free, HEPES-buffered media. The addition of 0.5 - 1 u M TTX had a minimal effect on resting pHj (pHj 7.40 ± 0.03 in the presence of TTX, n=\l; compared to pHj 7.44 ± 0.03 in the absence of TTX, «=28). In the presence of TTX, a 5 min period of anoxia induced an acidification of 0.10 ± 0.01 pH units that recovered by 0.02 ± 0.01 pH units, a 29 ± 8% recovery, in the continued absence of oxygen, followed by a post-anoxic alkalinization of 0.17 + 0.02 pH units (Fig. 20; Table 7). The magnitude of the anoxic acidification was reduced in the presence of TTX («=17; /?<0.05). In addition, the absolute magnitude of the per-anoxic rise in pHj was reduced in the presence of T T X although the relative magnitude of the per-anoxic recovery was not significantly different than observed in the absence of TTX (29 + 3% vs. 40 ± 5% recovery in the presence or absence of TTX, respectively) The fact that T T X was able to reduce the absolute magnitude of the intracellular acidification seen during anoxia suggested the possibility that T T X may help to maintain N a + / H + exchange activity during an anoxic period at a higher level than observed in the 70 absence of TTX. To investigate this possibility, in the presence of 0.5 - 1 u M TTX, intracellular acid loads were performed both before and during a transient anoxic period. In 7 cells examined, there was an overall 49 ± 3% decrease in the rate of pH; recovery from acid loads imposed during anoxia in the presence of T T X compared to the rate of pHj recovery established in the presence of TTX prior to anoxia in the same neurons (Fig. 21). The rates of recovery of pHj were plotted against the absolute pHj values at which they were measured and linear regressions were obtained relating the rates of pHj recovery to absolute pHj values under both normoxic (with TTX) and anoxic (with TTX) conditions. As previously seen in the parallel set of experiments conducted in the absence of T T X (see Section 5.2.3), in the presence of TTX, at every absolute value of pH i 5 the rate of pH; recovery was significantly slower during anoxia when compared to the corresponding pre-anoxic control rate of pH ; recovery established in the same cells (Fig. 22; p<0.05 at every pH; value). The slope of the linear regression line was reduced 3 1 3 1 from -27.96 x 10" s" under normoxic conditions to -18.59 x 10" s" under anoxic conditions, a 34% reduction in slope. In the absence of TTX, there was a similar 36% reduction in the slope of the linear regression line when comparing the normoxic and anoxic conditions. Moreover, in the presence of TTX, there was a shift of the x-intercept of the regression line in the acidic direction, from pHj = 7.50 under normoxic conditions to pH; = 7.42 under anoxic conditions. Thus, TTX failed to affect the reduction in the activity of the Na7H + exchanger evoked by a 5 min anoxic insult. Thus, while T T X reduced the magnitude of the intracellular acidification evoked by anoxia, it did not attenuate the degree of inhibition of Na7H + exchange evoked by anoxia. 71 5.4 The potential involvement of a voltage-activated H* conductance Anoxic depolarization occurs within the first few minutes of an anoxic insult (Balestrino, 1995; Roberts Jr. and Chih, 1997; Tanaka et al, 1997; Yamamoto et al, 1997). Given the contribution of voltage-sensitive proton conductances (gH+) to acid extrusion in a variety of cell types (reviewed by Lukacs et al, 1993; see Introduction, Section 4.3.2), the activation of a gH+ consequent upon anoxic depolarization may participate in the pHj response to anoxia. To examine the potential role of a gH+ in the pH, response to anoxia, experiments were performed in HC0 37C0 2-free, HEPES-buffered media containing 500 p M ZnCl 2 to block gH+. 500 p M ZnCl 2 had a minimal effect on steady-state pHj (pH; 7.36 ± 0.03 in the presence of ZnCl 2 , n-\l; compared with pH ( 7.30 + 0.04 in the absence of ZnCl 2 ; «=15; p>0.\). In response to a 10 min period of anoxia, the presence of 500 p M ZnCl 2 did not influence the magnitude of the internal acidification; however, the rise in pHj observed during anoxia was reduced from 0.08 ± 0.02 pH units (a 57 ± 11% recovery) under control conditions to 0.03 ± 0.01 pH units (a 26 ± 8% recovery) in the presence of ZnCl 2 (Fig. 23; Table 7; p<0.05). The presence of ZnCl 2 also significantly attenuated the magnitude of the post-anoxic alkalinization, as well as decreasing the time required to reach the maximum alkalinization (p<0.05 in each case; Table 7). These experiments suggest a role for a Zn2+-sensitive, gH+ in the production of the per-anoxic rise in pHj and the post-anoxic internal alkalinization observed in response to a 10 min period of anoxia. 72 6 The effect of changing ambient temperature The activity of the Na7H + exchanger in rat hippocampal neurons is temperature- sensitive (Baxter and Church, 1996; also see Bookstein et al, 1997; Marjanvoic et al, 1998 for evidence in other N H E isoforms). Furthermore, reductions in temperature are known to exert a neuroprotective action (Young et al, 1983; Kurihara et al, 1996; Kozlowski et al, 1997). To investigate whether the pHj response to anoxia was influenced by a decrease in temperature, 5 min periods of anoxia were imposed at 22°C rather than at 37°C. Prior to anoxia, in HC0 37C0 2-free, HEPES-buffered media at pH 7.35, steady- state pHj values were distributed, in a Gaussian manner, around a mean of pHj 7.23 ± 0.02 (range pHj 7.00 - 7.45; «=26; Fig. 24A). In HC0 37C0 2-buffered media, steady-state pH; values were similarly distributed, around a mean of pH; 7.24 ± 0.02 (range pH; 7.08 - 7.35; «=16; Fig. 24B). Under both buffering conditions, the mean steady-state pH; values were statistically lower at 22°C than the respective values observed at 37°C, suggesting a decrease in the relative importance of the Na7H + exchanger to the maintenance of steady- state pHi at 22°C (see Baxter and Church, 1996). 6.1 Steady state changes in intracellular pH evoked by anoxia at 22°C under HCO{/C02- free, HEPES-buffered conditions In HC0 37C0 2-free, HEPES-buffered media at 22°C, a 5 min period of anoxia induced an intracellular acidification of 0.10 ± 0.01 pH units («=9), that reached its maximum at 2.8 ± 0.3 min following the induction of anoxia (Fig. 25A; Table 8). In the 73 continued absence of oxygen, there was a 0.005 ± 0.002 pH unit increase of pH i ? a 4 ± 3% recovery. Upon the return to normoxia, an intracellular alkalinization of 0.04 ± 0.02 pH ; units occurred, which reached a maximum value after 7.2 +1.0 min (Table 8). Thus, the magnitude of each phase of the pHj response to anoxia was significantly reduced at 22°C when compared to the pH; response to 5 min periods of anoxia imposed under identical buffering conditions at 37°C (/?<0.05 in each case). 6.2 Steady state changes in intracellular pH evoked by anoxia at 22°C under HCO{/C02- buffered conditions Parallel experiments to those reported in Section 6.1 were performed under HC0 3 ~ /C02-buffered conditions at 22°C (Fig. 25B; Table 8). Under these conditions, a 5 min period of anoxia induced a maximum intracellular acidification of 0.057 ± 0.005 pH units (n=\3). During the continued absence of oxygen, pHj increased by 0.03 ± 0.01 pH units, a 53 ± 15% recovery. Upon the return to normoxia, a maximum intracellular alkalinization of 0.06 ± 0.01 pH units occurred, which was reached after 3.6 ± 0.6 min following the return to normoxia. As was the case under HC0 37C0 2-free, HEPES- buffered conditions, the magnitudes of all three phases of the pHj response to anoxia were significantly reduced when compared to the corresponding values obtained at 37°C (p<0.05 in each case). At 22°C, the magnitudes of the intracellular acidification observed during the anoxic period and the per-anoxic rise in pHj were significantly smaller and larger, respectively, under HC0 37C0 2-buffered conditions when compared with H C 0 3 7 C 0 2 - 74 free, HEPES-buffered conditions (Table 8). However, the magnitude of the post-anoxic alkalinization was not influenced by the presence or absence of H C 0 3 " at 22°C. 6.3 Anoxia-evoked changes in the activity of the Na+/H* exchanger at 22°C In HC0 3 /C0 2 -free, HEPES-buffered media, intracellular acid loads were performed to assess whether the decrease in the magnitude of the post-anoxic alkalinization observed at 22°C reflected a relative reduction in the activity of the N a + / H + exchanger following a 5 min period of anoxia at 22°C compared to 37°C. In 11 cells, there was a 102 ± 11% increase in the overall rate of pHj recovery from intracellular acid loads imposed following anoxia at 22°C (Fig. 26). Rates of pH ; recovery were plotted against absolute pH, values and linear regression lines relating rates of pH, recovery to absolute pH ; values were obtained under both pre-anoxic and post-anoxic conditions at 22°C. In a manner similar to that observed at 37°C (see Section 5.2.3), at every pHj value, the rate of pHj recovery was significantly faster after a 5 min period of anoxia compared to the pre-anoxic control rate of pHj recovery established in the same cells (Fig. 27; p<0.05 at every pH s value). The slope of the linear regression line relating pH ; to rates of 3 1 pHi recovery was increased from -0.76 x 10" s" under normoxic conditions at 22°C to 3 1 -1.47 x 10" s" under post-anoxic conditions, indicating that there was a post-anoxic stimulation of Na7H + exchange activity. Most importantly, at every absolute value of pH i 5 under both normoxic and post-anoxic conditions, rates of recovery of pHj from imposed acid loads were significantly faster at 37°C than at 22°C (Fig. 27; /?<0.05 at every pHj value under both normoxic and post-anoxic conditions). The results suggest 75 that while the activity of the N a + / H + exchanger is stimulated following transient periods of anoxia at both 22°C and at 37°C, the absolute level of N a + / H + exchange activity is lower at 22°C compared to 37°C. Thus, the reduced activity of the Na7H + exchanger likely underlies the reduced magnitude of the post-anoxic steady-state rise in pH; observed at 22°C. 7 The effects of changing extracellular pH In vivo ischemia and anoxia produce changes in extracellular pH (pH0) (Mutch and Hansen, 1984; Silver and Erecinska, 1992). In turn, p H 0 is known to influence pH; in rat hippocampal neurons (Church et al, 1998), possibly by altering the activity of the N a + / H + exchanger (Aronson et al, 1983; Jean et al, 1985; Vaughan-Jones and Wu, 1990). Thus, the effects of changing pH 0 from 7.35 to either 6.60 or 7.60 on the pHi response to a 5 min period of anoxia were examined. 7.1 The effects of reducing extracellular pH Exposure to a HC0 37C0 2-free, HEPES-buffered solution at pH 0 6.60 caused a rapid 0.49 ± 0.03 pH unit fall in pH ; (w=26). Once the new steady-state pH; level was reached, a 5 min period of anoxia induced an acidification of 0.15 ± 0.01 pH units (n=l0) that recovered by 0.02 ± 0.01 pH units, a 17 ± 9% recovery, within the anoxic period (Fig. 28; Table 9). Upon the return to normoxia, there was an internal alkalinization of 0.027 ± 0.005 pH units above the pre-anoxic steady-state level that reached its maximum after 4.0 ± 0.4 min. Compared to the results obtained at pH 0 7.35, the magnitude of the 76 anoxic acidification was not influenced by the reduction in pH 0 ; however, the magnitudes of the per-anoxic rise in pHj and the post-anoxic alkalinization were significantly smaller at pH 0 6.60 than observed at p H 0 7.35 (p<0.05 in each case). In addition, the time required to reach the maximum of the post-anoxic alkalinization was significantly reduced at pH 0 6.60 compared to pH„ 7.35. In many cell types, a reduction in pH 0 inhibits the activity of the N a + / H + exchanger (Jean et al, 1985; Vaughan-Jones and Wu, 1990). To investigate whether the attenuation of the post-anoxic alkalinization observed under pH 0 6.60 conditions was due to a relative decrease in the activity of the N a + / H + exchanger, intracellular acid loads were performed both before and after a 5 min period of anoxia at p H 0 6.60. A 5 min period of anoxia evoked a 173 + 16% increase in the overall rate of pH s recovery in the immediate post-anoxic period (Fig. 29; n=9). Rates of pHj recovery were plotted against absolute pHj values and linear regression lines relating the rates of pH; recovery and absolute pH ; values were obtained for the pre- and post-anoxic conditions. As was the case at pH 0 7.35, at pH 0 6.60 the rate of recovery of pHj was significantly faster following a 5 min i period of anoxia when compared with the corresponding pre-anoxic control rate of p r l recovery (Fig. 30; /?<0.05 at all pH; values except pH> 6.70). The slope of the linear regression line relating pHj to rates of pH; recovery was increased from -6.00 x 10"3 s"1 under pre-anoxic conditions to -19.21 x 10"3 s"1 in the post-anoxic period, indicating that an increase in the activity of the Na7H + exchanger occurs following anoxia under pH 0 6.60 conditions. On the other hand, anoxia did not influence the x-intercept of the linear regression lines relating rates of pH; recovery to absolute values of pHj. Thus, following 77 an anoxic period imposed at pH 0 6.60, N a + / H + exchange activity is stimulated, but there is no apparent change in the pH; sensitivity of the exchanger. At every pH; value, under both pre- and post-anoxic conditions, the rates of pH; recovery from imposed acid loads were significantly slower at pH 0 6.60 when compared with the corresponding rates of pHi recovery obtained prior to and following anoxia at pH 0 7.35 (Fig. 30). Thus, although NaTFT exchange activity is stimulated following anoxia at pH 0 6.60, the reduced magnitude of the intracellular post-anoxic alkalinization at pH 0 6.60 can be attributed to the reduced absolute level of N a + / H + exchange activity in the presence of an external acidosis. 7.2 The effects of increasing extracellular pH The next series of experiments were performed at p H 0 7.60. Exposure to a p H 0 7.60, HC0 37C0 2-free, HEPES-buffered solution caused a rapid 0.32 ± 0.01 pH unit increase in pHj (n=29). Once pH; had reached a new steady-state level, a 5 min period of anoxia induced an intracellular acidification of 0.03 ± 0.05 pH units («=10) that recovered by 0.08 ± 0.02 pH units, a 269 ± 80% recovery, in the continued absence of oxygen (Fig. 31; Table 9). Upon the return to normoxia, there was a maximum intracellular alkalinization of 0.28 ± 0.04 pH units which was attained after 3.8 ± 0.2 min upon the return to normoxia. The magnitude of the intracellular per-anoxic acidification was significantly smaller, whereas the magnitudes of the per-anoxic recovery of pH; and the post-anoxic alkalinization were significantly larger, than those observed at p H 0 7.35 (p<0.05 in each case). The time required to reach the maximum post-anoxic 78 alkalinization was significantly reduced at pH 0 7.60 compared with pH 0 7.35 (p<0.05; Table 9). To investigate whether the increase in the magnitude of the post-anoxic alkalinization observed under pH„ 7.60, compared to pH 0 7.35, conditions reflected a relative increase in the activity of the Na7H + exchanger, intracellular acid loads were performed before and after a 5 min period of anoxia at p H 0 7.60. Following anoxia, in 7 cells, there was a 127 ± 22% increase in the overall rate of pHj recovery from acid loads imposed at pH 0 7.60 (Fig. 32). Rates of recovery of pHj were plotted against the pH f values at which they were measured, and linear regression lines relating the rates of pH ; recovery and absolute pHj values were obtained for the normoxic and post-anoxic conditions. At pH 0 7.60, the rate of recovery of pH, was significantly faster following a 5 min period of anoxia when compared to the corresponding pre-anoxic control rates of pH; recovery (Fig. 33; p<0.05 at all pHj values except pHj 7.20). The slope of the linear regression line relating absolute values of pHi to rates of pHj recovery was increased from 3 1 3 1 -20.12 x l 0 " J s under pre-anoxic control conditions to -22.01 x 10" s" following a 5 min period of anoxia. In addition, there was an alkaline shift in the pHj sensitivity of the N a + / H + exchanger, as indicated by a shift in the x-intercept of the regression line from pH; = 7.94 to pH ; = 8.39 under post-anoxic conditions. As seen at pH 0 7.35 and at pH„ 6.60, under pH 0 7.60 conditions, the activity of the NaVFf exchanger is stimulated following a transient anoxic period. Furthermore, the rates of pH; recovery were significantly faster at pH 0 7.60 when compared to the corresponding rates of pH ; recovery obtained both prior to and following an anoxic period 79 at pH 0 7.35 (Fig. 33). Thus, the relatively greater activity of the Na7H + exchanger after anoxia at pH 0 7.60 compared to pH 0 7.35 likely underlies the increased magnitude of the post-anoxic alkalinization observed following an anoxic period at pH 0 7.60. 7.3 The dependence of intracellular pH on extracellular pH under normoxic conditions In the absence of an anoxic insult, rates of pHi recovery following intracellular acid loads imposed at pH 0 6.60 were significantly slower, and the rates of pHj recovery at pH 0 7.60 significantly faster, when compared to the corresponding rates of pHj recovery observed at pH 0 7.35. Thus, as pH 0 increases, there was a progressive increase in the slope of the linear regression lines relating rates of pH| recovery to absolute pH, values (the slopes being -6.00 x IO"3 s 1, -17.73 x IO"3 s"1 and -20.12 x 10~3 s"1 at pH„ 6.60, 7.35 and 7.60, respectively). Similarly, there was a progressive alkaline shift in the x-intercept of the linear regression lines as pH 0 increased (the x-intercepts being 6.91, 7.42, and 7.94 at pH 0 6.60, 7.35 and 7.60, respectively). Taken together, these data indicate that NaTFf exchange in rat hippocampal neurons is inhibited at acidic pH 0 values and stimulated at alkaline pH 0 values. The dependence of NaTFf exchange activity on pH 0 may contribute to the overall p H 0 dependence of pH; in post-natal rat hippocampal neurons (see Church et al, 1998). To examine the relationship between pH 0 and pH, in adult rat hippocampal neurons, cells were exposed to HC0 37C0 2-free, HEPES-buffered media at pH 7.75, 7.60, 7.35, 6.80 or 6.60. Exposure to pH 7.75 and pH 7.60 media caused 0.40 + 0.03 (n=5) and 0.32 ± 0.01 (n=29) pH unit increases, respectively, in steady-state pHj. Exposure to pH 6.80 and 6.60 80 media caused 0.42 ± 0.04 (n=2) and 0.49 ± 0.03 (n=26) pH unit decreases, respectively, in steady-state pH;. The new steady-state pH; was then plotted against pH 0 . Thus, in contrast to peripheral cell types but in agreement with data obtained from cultured post- natal rat hippocampal neurons, in adult rat hippocampal neurons there was a significant dependence of pH, on pH 0 (Fig. 34; ApHj:ApH 0 = 0.74; r2=0.98). Taken together, the above data support the notion that p H 0 is capable of influencing pH ; in adult rat hippocampal CA1 neurons by altering NaTFT exchange activity. 8 The potential contribution of changes in intracellular buffering power The results presented above indicate that transient anoxic insults lead to changes in the activity of the Na7H + exchanger in rat hippocampal neurons. However, it is established that changes in intracellular buffering power may contribute to changes in the rate of acid extrusion (Boron, 1989). For example, exposure of cardiac Purkinje fibres to the aerobic inhibitor, cyanide, caused a 47% decrease in the rate of pHj recovery from imposed intracellular acid loads that was largely attributed to an increase in intracellular buffering power (Wu and Vaughan-Jones, 1994). Exposure to the weak base, NH 4 C1, causes an intracellular alkalinization, the magnitude of which, in the absence of C 0 2 , provides a reasonable estimation of the intrinsic buffering power (Pi) of the cell (Amos and Richards, 1996; Baxter and Church, 1996; Smith et al, 1998). The magnitudes of the increase in pH ; evoked by 40 m M NH 4C1 were similar under pre-anoxic, anoxic and post-anoxic conditions (0.27 ± 0.05 pH units, 0.21 ± 0.04 pH units, and 0.21 ± 0.02 pH units, respectively ; n>9 in each case). In 81 addition, the presence of TTX failed to affect the magnitude of this alkalinization both under normoxic and anoxic conditions (a 0.27 ± 0.08 pH unit vs. 0.25 ± 0.03 pH unit increase under normoxic and anoxic conditions, respectively; n=l); thus, the presence of T T X failed to affect significantly % Moreover, it has been shown in hippocampal neurons that perfusion with a Na+-free solution does not alter P, (Amos and Richards, 1996). Therefore, at pH 0 7.35, a change in p; appears unlikely to be able to account for the marked changes in the rates of acid extrusion seen either during or following an anoxic period. It is established that Pi in rat hippocampal neurons increases with a decrease in pHj (Bevensee et al, 1996). In the present study, the magnitudes of the pre- and post- anoxic alkalinizations produced upon exposure to NFLCl were significantly smaller at pH 0 6.60 and significantly larger at pH„ 7.60 than the increases in pH ; observed at pH 0 7.35. Thus, under pre-anoxic conditions, the increase in pH, evoked by 40 m M NH 4C1 was 0.17 ± 0.02 pH units at pH 0 6.60 and 0.43 ± 0.04 pH units at pH 0 7.60 (n>l 1 in each case; p<0.05 for corresponding change measured under pH 0 7.35 conditions). In the post- anoxic period, the increase in p l i was 0.14 ± 0.02 pH units at pH 0 6.60 and 0.45 ± 0.08 pH units at pH 0 7.60 (n>ll in each case; /?<0.05 for corresponding change measured at pH 0 7.35). These data confirm the inverse relationship between p ; and pHj in rat' hippocampal neurons and suggest the possibility that changes in p( may contribute to the differences in the rates of acid extrusion observed at pH 0 6.60 and 7.60, when compared to the rates of acid extrusion at pH 0 7.35. However, at a given pH„ value, there was no change in the magnitude of the increase in pHj when comparing the pre- and post-anoxic 82 values. Thus, at each of the pH 0 conditions tested, a change in ft, in the post-anoxic period cannot account for the increase in acid extrusion rate observed following a transient period of anoxia. 9 The Role of Extracellular Glucose Concentration Acutely dissociated neurons are commonly prepared in and exposed to solutions containing 17.5 m M glucose, a concentration which, while preserving the integrity of these isolated cells in vitro (e.g. Fan and Wong, 1996; Dzoljic et al, 1998), is considered hyperglycemic in vivo (Ibayashi et al, 1986, Silver and Erecinska, 1994). In addition, it is established that extracellular [glucose] affects the magnitude of the internal acidification seen during in vivo anoxia/ischemia, at least in part by determining the internal lactate and ATP levels during and following the insult (Ljunggren et al, 1974; Katsura et al, 1992a; but see Hope et al, 1988; Tyson et al, 1993). Thus, the pHj response to a 5 min period of anoxia was assessed after reducing the extracellular glucose concentration from 17.5 m M to 5.0 mM, while maintaining the osmolality of the solution with the addition of 12.5 m M D-mannitol (Morimoto et al, 1994; Cendoroglo et al, 1998; Wang et al, 1998). The osmolality of the HC0 37C0 2-free, HEPES-buffered media containing 17.5 m M glucose was 295 ± 2 mOsm/kg H 2 0 (n=4) and the osmolality of the HEPES-buffered solution containing 5.0 m M glucose was 296 ± 2 mOsm/kg H 2 0 («=4), a value not statistically different from the former. In the presence of 5.0 m M glucose, a 5 min anoxic period induced an intracellular acidification of 0.12 + 0.01 pH units that reached its maximum 2.3 ± 0.2 min following 83 the start of anoxia (Fig. 35B; Table 10; n=22). In the continued absence of oxygen, pHj recovered by 0.021 ± 0.005 pH units, a 21 ± 6% recovery. Upon the return to normoxia, there was an intracellular alkaline shift of 0.18 ± 0.02 pH units, reaching a maximum after 8.9 ± 0.5 min after the end of the anoxic period. As was observed under 17.5 m M glucose conditions, the initial steady-state pH, was positively correlated with the magnitude of the anoxic acidification (^=0.73). Changing the duration of the anoxic from 5 min to either 3 or 10 min did not significantly influence the magnitudes of the intracellular acidification seen during anoxia or the intracellular alkalinization observed following anoxia (Figs. 35A and 35C; Table 10). However, the magnitude of the per-anoxic rise in pHj was significantly larger during a 10 min period of anoxia than during a 3 min period of anoxia (p<0.05). The pH; response to an anoxic period imposed during perfusion with media containing 5.0 m M glucose was qualitatively similar to that observed in the presence of 17.5 m M glucose. However, various phases of the pH, response were influenced by the change in extracellular glucose concentration. Firstly, the magnitudes of the anoxic acidifications evoked by a 5 or 10 min period of anoxia were statistically smaller in the presence of 5.0 m M glucose when compared to those observed in the presence of 17.5 m M glucose (p<0.05 in each case). Secondly, the magnitude of the per-anoxic rise in pHj was statistically smaller during 3 or 5 min periods of anoxia imposed in the presence of 5.0 m M glucose, despite the fact that there were no differences in the times to the start of these increases in pH ; (which occurred at 3.6 ± 0.2 min and 3.1 ± 0 . 2 min following the start of anoxia in 5.0 and 17.5 m M glucose, respectively; ri>\\; /?<0.05 in each case). 84 Finally, the magnitude of the post-anoxic alkalinization following a 10 min period of anoxia was significantly larger in the presence of 17.5 m M glucose compared to 5.0 m M glucose. 9.1 Role of the Na+/H¥ exchanger at a reduced extracellular glucose concentration To investigate the potential role of the Na7H + exchanger in the presence of 5.0 m M glucose, a 5 min period of anoxia was induced under reduced-Na+, N M D G + - substituted conditions. Exposure to this solution caused a rapid intracellular acidification of 0.45 ± 0.04 pH units that recovered to a plateau value of 101 ± 1% of the initial pHj value after 15 + 2 min (n=17; Fig. 36A). A l l parameters of this recovery of pHj were not statistically different than found during perfusion with a reduced-Na+, 17.5 m M glucose solution (see Section 5.2.1). During perfusion with a reduced-Na+, 5.0 m M glucose-containing medium, a 5 min anoxic period evoked an intracellular acidification of 0.09 ± 0.02 pH units (n-\l) that recovered by 0.02 + 0.01 pH units, a 17 ± 8% recovery, in the continued absence of oxygen (Fig. 36A; Table 11). Upon the return to normoxia, there was an intracellular alkalinization of 0.10 + 0.03 pH units. While the magnitudes of the per-anoxic acidification and the per-anoxic rise in pH; were not influenced by reduced-Na+, N M D G + - substituted conditions, the magnitude of the post-anoxic alkalinization was significantly reduced. This finding parallels the observation made with 17.5 m M glucose (Table 6) and is consistent with the possibility that an increase in the activity of the N a + / H + 85 exchanger is responsible for the production of the post-anoxic alkalinization in the presence of 5 m M as well as 17.5 m M glucose. Experiments were then performed in a reduced-Na+, Li+-substituted medium containing 5.0 m M glucose. The exposure to this solution caused an intracellular acidification of 0.30 ± 0.03 pH units that recovered to 102 ± 1% of the initial steady-state pHj value over 18 + 1 min (n=\5; Fig. 37B). A 5 min period of anoxia induced an intracellular acidification that recovered slightly in the continued absence of oxygen, and upon the return to normoxia, there was a rapid intracellular alkalinization (Fig. 36B; Table 11). Under Li+-substituted conditions, the magnitude of the post-anoxic alkalinization was not significantly different than observed in the presence of normal extracellular N a + and was significantly larger than seen under NMDG+-substituted conditions (Table 11; p<0.05 in each case). In addition, as observed in the presence of 17.5 m M glucose, the time required to reach the maximum of the post-anoxic alkalinization was significantly faster under reduced-Na+, Li+-substituted conditions than observed in the presence of normal extracellular Na + . However, analogous to the situation in the presence of 17.5 m M glucose, the restoration of the post-anoxic alkalinization was not secondary to a change in its time course, as the magnitude of the post-anoxic alkalinization was measured at fixed time points following a 5 min period of anoxia and this possibility was found not to be the case (data not shown). Taken together, these experiments suggest that, in the presence of 5.0 m M glucose, the N a + / H + exchanger is involved in the production of the post-anoxic alkalinization. Thus, the activity of the N a + / H + exchanger appears to be influenced to only a minor degree by 86 relatively limited changes in extracellular glucose concentration. On the other hand, at both 5.0 m M and 17.5 m M glucose, a reduction in Po2 has a significant influence on Na7H + exchange activity in the post-anoxic period. 10 The potential role of intracellular second messenger systems These studies described above establish that a change in the activity of the Na7H + exchanger contributes to the pHj response to a transient period of anoxia in adult rat hippocampal neurons. In peripheral cell types, the post-anoxic increase in Na7H + exchange activity is consequent upon the normalization of pH 0 . The fall in pH 0 during anoxia/ischemia is suggested to inhibit Na7H + exchange, which can only respond to the fall in pH ; induced by ischemia once pH 0 returns to normal. In contrast to these data, in the present study, given that pH 0 was held constant throughout the anoxic and post-anoxic periods, alternate factors must contribute to the increase in the activity of the Na7H + exchanger following transient periods of anoxia. Thus, preliminary experiments investigated the potential role of anoxia-evoked changes in second messenger systems in producing the observed changes in Na7H + exchange activity. The present focus was on the involvement of the c A M P / P K A pathway because: i) it has been shown to be involved in the regulation of Na7H + exchange activity in acutely dissociated adult rat hippocampal CA1 neurons (Smith et al, 1998); and ii) its activity is altered during and following transient periods of anoxia (Kobayashi et al, 1977; Whittingham et al, 1984; Blomqvist et al, 1985; Domanska-Janik and Pylova, 1989). 87 The application of 50 p M Rp-cAMPS, a selective inhibitor of P K A (Parker- Botelho et al, 1988), did not influence pPf In the presence of Rp-cAMPS, a 5 min period of anoxia induced an intracellular acidification which was statistically smaller than seen in the absence of Rp-cAMPS (p<0.05; n=A; Table 12). In addition, upon the return to normoxia, the presence of Rp-cAMPS reduced the magnitude post-anoxic alkalinization (p<0.05), suggesting that the increase in [cAMP]j known to occur in the immediate post- anoxic period in rat hippocampal neurons may act via P K A to promote an increase in the activity of the NaTFT exchanger following a transient period of anoxia. Both 10 p M noradrenaline and 10 p M isoproterenol stimulate NaTFf exchange activity in rat hippocampal neurons via p-adrenoceptors and the c A M P / P K A pathway (Smith et al, 1998). In the present study, application of 10 p M noradrenaline increased steady-state pPf, by 0.15 ± 0.03 pH units («=5). Similarly, the application of 10 p M isoproterenol increased steady-state pHj by 0.22 ± 0.09 pH units (n=5). Given that these treatments activate NaTFT exchange to a similar extent through the same intracellular pathway (Smith et al, 1998), their effects on the pPf response to transient anoxic periods were pooled. Thus, once the new steady-state pH; was reached in the presence of a p- adrenoceptor agonist, a 5 min period of anoxia induced an intracellular acidification that recovered slightly in the continued absence of oxygen (Table 12). Upon the return to normoxia, there was an intracellular alkalinization, the magnitude of which was significantly larger in the presence of a p-adrenoceptor agonist than observed in its absence (n=10; /><0.05). The time required to reach the maximum post-anoxic alkalinization was significantly faster in the presence of a P-adrenoceptor agonist than in 88 its absence. Thus, inhibiting the c A M P / P K A pathway reduces, and stimulating the c A M P / P K A pathway increases, the magnitude of the post-anoxic alkalinization, a result consistent with the involvement of the Na7H + exchanger in the production of the post- anoxic increase in pH;. 11 Intracellular pH regulation in the absence of extracellular sodium A n observation made during the course of this study was the recovery of pH s from the intracellular acidification produced upon exposure to zero-Na+ or reduced-Na+, H C 0 3 ' /C02-free, HEPES-buffered solutions, media which block the Na7H + exchanger as well as all HC03"-dependent pH, regulating mechanisms. A similar finding has been reported by others in hippocampal neurons (Bevensee et al, 1996). In a concerted effort to undercover the mechanism(s) underlying this response, the effects of a number of pharmacological treatments and ionic substitutions on this alkalinizing process were tested (see Table 13). Pharmacological treatments included 50 p M omeprazole (an inhibitor of P-type ATPases), 1-2 p M bafilomycin A ! (an inhibitor of V-type ATPases), 300 u M eosin Y (an inhibitor of Ca 2 7H + ATPases), 500 u M ZnCl 2 (an inhibitor of gH+), and 5 m M 4-a-5-hydroxycinnamate (an inhibitor of lactate/H+ cotransport). Ionic manipulations included the removal of extracellular K + , Ca , CI" (with NaCl, KC1, and CaCl 2 being substituted with their respective gluconate salts), or M g 2 + , which act to inhibit K + / H + exchange, C a 2 + / H + exchange, Na+-independent H+/C1" exchange, and ATPase activity. The parameters measured to assess the influence of these maneuvers on the recovery of pH, in the absence of extracellular N a + were: i) the 89 magnitude of the initial fall in pH; upon exposure to zero-Na+ media; ii) the time, from the point of exposure to zero-Na+ media, to the initiation of the rise in pH^; and iii) the time required, from the point of exposure to zero-Na+ media, to reach a plateau during the continued perfusion with zero-Na+ media. None of these manipulations influenced significantly steady-state initial pH ( (see Table 13). Moreover, none of these manipulations completely blocked the prf, recovery observed during perfusion with zero-Na+ media. However, perfusion with 500 u M ZnCl 2 , a [K+]0-free, or a [Ca2+]0-free solution significantly delayed the onset of pH ( recovery. While these experiments suggest the presence of (an) alternate pHj regulating mechanism(s) in acutely dissociated adult rat hippocampal C A T neurons, the identity of the mechanism(s) which participate in the observed internal alkalinization produced under conditions where the Na7H + exchanger and HC03"-dependent mechanisms are blocked remains unknown. The possibility also remains that this mechanism(s) is not functional under resting conditions and is activated only during perfusion with a zero-Na+ solution. 90 Table 4: Magnitude and time course of anoxia-evoked changes in intracellular pH under HC03"-free, HEPES-buffered conditions Duration of Anoxia 3 minutes 5 minutes 10 minutes (n=13) («=28) (n=15) Magnitude of (pH; units): Acidic Shift 0.17 ± 0.02c 0.16 ± 0.01 0.15 ± 0.01 Recovery 0.015 ± O ^ 3 - " 0.05 + 0.01 0 .08±0.02 a Alkaline Shift 0.19 ± 0.05b 0.20 ± 0.02 0.27 ± 0.04^ Time to Reach (minutes): Start of Acidification 0.9 ±0.1 1.0 + 0.1 1.1 ± 0.2 Peak Acidification 1.9±0.2 a- b 2.5 ± 0.2 2.6 ±0 .3 Peak Alkalinization 11.6 ± 1.9 9.5 ±1.1 9.2 ± 1.5° a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC0 3 '-free, HEPES-buffered solution. b indicates statistical significance when compared to the equivalent parameter after 10 min of anoxia in a HC03"-free, HEPES-buffered solution. c indicates statistical significance when compared to the equivalent parameter in a HC0 37C0 2-buffered medium 91 Table 5: Magnitude and time course of anoxia-evoked changes in intracellular pH under HC0 37C0 2-buffered conditions Duration of Anoxia 3 minutes 5 minutes 10 minutes (n=17) (w=14) («=12) Magnitude of (pH; units): Acidic Shift 0.12± 0.0rc 0.18± 0.02 0.15 ± 0.02 Recovery 0.02±0.0rb 0.07±0.01b 0.11 ±0.02 Alkaline Shift 0.13 ± 0.02 0.16 ±0.05 0.19 ± 0.02c Time to Reach (minutes): Start of Acidification 0.9 ± 0.1 1.1 ±0.1 0.9 ± 0.1 Peak Acidification 1.9 ± 0.1b 2.2 ±0.2 2.5 ±0.3 Peak Alkalinization 11.9 ±1 .6 10.8 ± 1.3 13.0±0.8 C a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC0 37C0 2-buffered medium b indicates statistical significance when compared to the equivalent parameter after 10 min of anoxia in a HC0 37C0 2-buffered medium c indicates statistical significance when compared to the equivalent parameter in a HEPES-buffered medium 92 Table 6: The influence of the reduction of extracellular Na on the pHj response to anoxia under HC03"-free, HEPES-buffered conditions Ionic Substitution Control Low Na + Low Na + (NMDG + ) (Li +) («=28) (n=20) («=13) Magnitude of (pH; units): Acidic Shift 0.16 ± 0.01 0.11 ± 0 . 0 1 " 0 . 1 8 ± 0 . 0 2 b Recovery 0.05 ±0.01 0.04 ±0.01 0.05 ±0.01 Alkaline Shift 0.20 ± 0.02 0.12 ± 0.02a 0.20 ± 0.03" Time to Reach (minutes): Start of Acidification 1.0 ± 0.1 1.1 +0.1 1.1 ±0.1 Peak Acidification 2.5 ± 0.2 2.8 ± 0.2 2.6 ±0 .3 Peak Alkalinization 9.5 ± 1.1 6.4 ± 0.8" 6.5 ± 0.5a a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC03'-free, HEPES-buffered solution containing normal extracellular Na+. b indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a low-Na+, NMDG-substituted, HC03'-free, HEPES-buffered solution 93 Table 7: Magnitude and time course of anoxia-evoked changes in pH; in the presence of T T X o r Z n C l 2 Control 0.5 -1 u M Control 500 u M TTX ZnCl 2 («=28) (»=17) ("-15) ("=17) Duration of Anoxia: 5 min 5 min 10 min 10 min Buffer: HEPES HEPES HEPES HEPES Magnitude of (pH; units): Acidic Shift 0.16 ± 0.01 o.io ± o.or 0.15 ± 0.01 0.16 + 0.02 Recovery 0.05 ± 0.01 0.02±0.01 a 0.08 ± 0.02 0.03 ±0 .01 b Alkaline Shift 0.20 ± 0.02 0.17 ± 0.02 0.27 ± 0.04 0.13 ±0.02 b Time to Reach (min): Start of Acidification Peak Acidification Peak Alkalinization 1.0 ±0.1 1.1 ±0.1 2.5 ±0.2 2.1 ±0.1 9.5 ±1.1 8.3 ±1.0 1.1 ±0 .2 0.9 ±0.1 2.6 ±0.3 3.3 ±0.5 9.2 ±1.5 4.6 ±0.5" a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC03"-free, HEPES-buffered solution in the absence of any pharmacological treatment. b indicates statistical significance when compared to the equivalent parameter after 10 min of anoxia in a HC03"-free, HEPES-buffered solution in the absence of any pharmacological treatment. 94 Table 8: The influence of temperature on the magnitude and time course of anoxia- evoked changes in pHj 37°C 22°C 37°C 22°C (»=28) (n=9) (»=14) (n=l3) Buffer HEPES HEPES H C 0 3 7 C 0 2 H C 0 3 7 C 0 2 Magnitude of (pH, units): Acidic Shift 0.16 ± 0.01 0 .10±0.01 a 0.18± 0.02 0.057 ± 0.005bc Recovery 0.0510.01 0.005 ± 0.002a 0.07 + 0.01 0.03 + 0.01 b c Alkaline Shift 0.20 ± 0.02 0.04 ± 0.02a 0.16 ±0.05 0 .06±0 .01 b Time to Reach (minutes): Start of Acidification 1.0 ±0.1 1.0 ±0.2 1.1 ±0.1 0 . 5 ± 0 . 1 b c Peak Acidification 2.5 ±0.2 2.8 ± 0.3 2.2 ± 0.2 1.4 ± 0.1 b c Peak Alkalinization 9.5 ± 1.1 7.2 ± 1.0 10.8 ± 1.3 3.6 ± 0.6 b c a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC0 3 7C0 2 -free, HEPES-buffered solution at 37°C. b indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC0 37C0 2-buffered solution at 37°C. 0 indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC03"-free, HEPES-buffered solution at 22°C 95 Table 9: The influence of pH 0 on the magnitude and time course of anoxia-evoked changes in p r l Extracellular pH 7.35 6.60 7.60 (n=28) (/i=10) («=10) Buffer HEPES HEPES HEPES Magnitude of (pHj units): Acidic Shift 0.16 ± 0.01 0.15 + 0.01 0 .03±0.05 a Recovery 0.05 ±0.01 0 .02±0.01 a 0.08 ± 0.02a Alkaline Shift 0.20 ± 0.02 0.027 ±0.005 a 0.28 ± 0.04a Time to Reach (minutes): Start of Acidification 1.0 ±0.1 0.8 ± 0.2 2 .0±0 .2 a Peak Acidification 2.5 ± 0.2 2.8 ±0 .3 3.1 ± 0.2 Peak Alkalinization 9.5 ± 1.1 4.0 ± 0.4a 3.8 ± 0.2a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC03"-free, HEPES-buffered solution at p H 0 7.35. 96 Table 10: Magnitude and time course of anoxia-evoked changes in pHj under HC03"-free, HEPES-buffered conditions in the presence of 5.0 m M glucose Duration of Anoxia 3 minutes 5 minutes 10 minutes (w=10) (K=22) («=8) Magnitude of (pHj units): Acidic Shift 0.14 ± 0.02 0.12 ± 0.01" 0 . 1 1 ± 0 . 0 1 b Recovery 0.004 ± 0.002^ 0.021 ± 0.005b 0.05 ± 0.02 Alkaline Shift 0.10 ± 0.03 0.18 ± 0.02 0.13 ± 0.03b Time to Reach (minutes): Start of Acidification 0.9 ±0.1 0.8 ±0.1 1.0 ±0 .2 Peak Acidification 2.0 ± 0.2 2.3 ±0.2 2.4 ±0 .2 Peak Alkalinization 6 . 3 ± 0 . 8 a b 8.9 ±0.5 4.7 ± 0.8a-b a indicates statistical significance when compared to the equivalent parameter in response to a 5 min period of anoxia in a HC03"-free, HEPES-buffered medium containing 5.0 m M Glucose b indicates statistical significance when compared to the equivalent parameter in a HC03"-free, HEPES- buffered medium containing 17.5 m M Glucose (see Table 4) 97 Table 11: The influence of the reduction of extracellular N a + on the anoxia-evoked changes in pH; under HCCy-free, HEPES-buffered conditions in the presence of 5.0 m M glucose Ionic Substitution Control Low Na + Low Na + (NMDG + ) (Li +) («=22) (w=17) («=15) Magnitude of (pHj units): Acidic Shift 0.12 ± 0.01 0.09 ± 0.02 0.14 ± 0.02 Recovery 0.021 ±0.005 0.02 ±0.01 0.02 ±0.01 Alkaline Shift 0.18 ± 0.02 0.10 ± 0.03a 0.20 ± 0.02b Time to Reach (minutes): Start of Acidification 0.8 ±0.1 1.5 ± 0.3a 0 . 9 ± 0 . 1 b Peak Acidification 2.3 ± 0.2 3.1 ± 0.2a 1.8 ±0.2 Peak Alkalinization 8.9 ± 0.5 6.2 ± 0.8a 5 .8±0 .6 a a indicates statistical significance when compared to the equivalent parameter in a HC03"-free, HEPES- buffered medium containing 5.0 m M Glucose and normal extracellular Na + . b indicates statistical significance when compared to the equivalent parameter in a low Na + , N M D G + - substituted HC03~-free, HEPES buffered medium containing 5.0 m M Glucose. 98 Table 12: Magnitude and time course of anoxia-evoked changes in pH; in the presence of agents that influence the activity of the c A M P / P K A pathway Control 50 u M Rp-cAMPS 10 u M NA/ISO («=28) (/i=12) («=10) Buffer: HEPES HEPES HEPES Magnitude of (pH; units): Acidic Shift 0.16 ± 0.01 Recovery 0.05 ±0.01 Alkaline Shift 0.20 ± 0.02 0.07 ± 0.01" 0 .03±0.01 a 0.11 ± 0.03a 0.10 ± 0.02a 0.04 ± 0.02 0.27 ± 0.03a Time to Reach (minutes): Start of Acidification 1.0 ±0.1 Peak Acidification 2.5 ± 0.2 Peak Alkalinization 9.5 ± 1.1 1.1 ±0.2 2.3 ± 0 . 3 a 4 .5±0 .5 a 0.8 ± 0.2 2.2 ± 0.5 4.0 ± 0.5a a indicates statistical significance when compared to the equivalent parameter after 5 min of anoxia in a HC0 3 '-free, HEPES-buffered solution in the absence of any pharmacological treatment. Abbreviations: N A , Noradrenaline; ISO, Isoproterenol 99 Table 13: The influence of various pharmacological treatments on the recovery of pHj in the continued absence of extracellular Na + Parameters Measured in Na+-free Media Resting pHj Drop in pH ( Time to start of pHj recovery Time to reach a plateau n Control 7.29 ± 0.29 0.46 ± 0.03 4.5 ± 0.4 25.6 ±2 .1 36 2 u M Bafilomycin 7.30 ± 0.03 0.44 ± 0.04 3.0 ± 1.5 25.3 ±3 .8 8 0 C a 2 + 7.15 ±0.06 0.46 ± 0.04 7.6 ± 1.7' 46.6 ± 18.3a 10 5 m M a-CHC 7.20 ±0.10 0.46 ± 0.07 3.0 ±0 .5 11.2 ± 1.6a 6 ocr 7.08 ±0.15 0.42 ±0.13 6.5 ± 1.1 21.7 ± 5.8 5 300 u M Eosin Y 7.08 ± 0.08a 0.39 ±0.06 5.7 ± 1.2 14.3 ± 1.5a 10 5.0 m M Glucose 7.23 ±0.14 0.41 ±0.03 5.1 ±3 .0 16.1 ± 2 . 1 a 3 o r 7.15 ±0.08 0.66 ± 0.07a 7.1 ± 0 . 8 a 26.9 ±2 .1 21 0 M g 2 + 7.2 ± 0.34 0.38 ±0.04 4.7 ±0 .7 52.0 ± 13.2° 11 50 u M Omeprazole 7.16 ±0.3 0.32 ± 0.04 4.9 ±0 .7 27.5 ±1 .2 3 500 u M ZnCl 2 7.20 ±0.07 0.79 ± 0.05a 7.9 ± 0.6a 28.4 ±4 .2 10 indicates statistical significance when compared to the equivalent parameter observed in a Na -free, NMDG+-substituted, HEPES-buffered solution in the absence of any pharmacological treatment 100 Figure 6. Distributions of steady-state pH, values for all neurons during perfusion with HC037C02-free, HEPES-buffered and HC037C02-buffered media at 37°C A . The distribution of the resting pH ( values for 694 adult rat hippocampal CA1 neurons during perfusion with HC0 37C0 2-free, HEPES-buffered media at pH 7.35 (bin width = 0.05 pH units). The data were best fitted by a bimodal Gaussian distribution with means at pH; 6.93 ± 0.17 and 7.37 ± 0.14. B. The distribution of resting pHj values for 83 neurons during perfusion with HC0 37C0 2-buffered media (bin wadth = 0.05 pH units) was fitted with a Gaussian distribution around a mean of 7.30 ±0.17.  102 Figure 7. Effects of a 5 min period of anoxia on steady-state pli during perfusion with HC037COrfree, HEPES-buffered medium A . Upon exposure to a 5 min period of anoxia, the initial resting pHi value of ~ 7.26 fell ~ 0.14 pH units, reaching its maximum ~ 1 min following the induction of anoxia. pH ; recovered ~ 0.11 pH units (79%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.24 pH units above the initial resting pHj value, which reached its maximum ~ 7 min following the return to normoxia. B. The stability of the corresponding BI4S2 value, during the experiment depicted in Fig. 7A, indicates that the membrane integrity of the neuron was not compromised (see Materials and Methods, Section 3.2). Time (min) 104 Figure 8. Effects of a 3 and 10 min period of anoxia on steady-state pH; during perfusion with HC037C02-free, HEPES-buffered medium A. Upon exposure to a 3 min period of anoxia, the initial resting pH| value of ~ 7.26 fell -0 .10 pH units, reaching its maximum ~1.7 min following the induction of anoxia. pHj recovered by - 0.02 pH units (20%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of - 0.16 pH units above the initial resting pHj value, which reached its maximum at - 8.7 min following the return to normoxia. B. Upon exposure to a 10 min period of anoxia, the initial resting pHj value of - 7.10 fell - 0.18 pH units, reaching its maximum - 4.4 min following the induction of anoxia. pH ; recovered - 0.19 pH units (106%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of - 0.38 pH units above the initial resting pHj value, which reached its maximum at - 9.1 min following the return to normoxia. Each trace represents data collected from a different neuron. Time (min) 106 Figure 9. Effects of a 3, 5 and 10 min period of anoxia on steady-state pH, during perfusion with HC037C02-buffered medium A. Upon exposure to a 3 min period of anoxia, the initial resting pH, value of ~ 7.35 fell ~ 0.14 pH units, reaching its maximum at ~ 1.8 min following the induction of anoxia. pH; did not rise during the anoxic period. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.12 pH units above the initial resting pH; value, which reached its maximum at ~ 7.6 min following the return to normoxia. B. Upon exposure to a 5 min period of anoxia, the initial resting pH, value of ~ 7.14 fell ~ 0.14 pH units, reaching its maximum at ~ 1.9 min following the induction of anoxia. pH ; recovered by ~ 0.08 pH units (57%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.22 pH units above the initial resting pH; value, which reached its maximum at ~ 7.5 min following the return to normoxia. C. Upon exposure to a 10 min period of anoxia, the initial resting pH; value of ~ 7.30 fell ~ 0.15 pH units, reaching its maximum at ~ 1.2 min following the induction of anoxia. pHj recovered ~ 0.15 pH units (100%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.21 pH units above the initial resting pH; value, which reached its maximum at ~ 4.5 min following the return to normoxia. Each trace represents data collected from a different neuron. 107 Time (min) 108 Figure 10. Effects of a 5 min period of anoxia on BI3i4/BI3S0 ratio values The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. The resting BI334/BI3g0 ratio value (the ratio of the background-corrected I334 and 7 3 8 0 ) was ~ 0.5. Approximately 2 min following the start of a 5 min period of anoxia, the ratio value increased to ~ 2.5, reaching its maximum value ~ 3.5 min following the induction of anoxia. Upon the return to normoxia, the BI334/BI3S0 ratio value remained elevated. 109 110 Figure 11. Effects of a 5 min exposure to HC037C02-buffered medium equilibrated with 95% Ai7 5% CO, for > 18 hours on steady-state pH, Upon exposure to a 5 min period of anoxia, the initial resting pHj value of ~ 7.38 fell ~ 0.13 pH units, reaching its maximum ~ 2 min following the induction of anoxia. pH; recovered by ~ 0.07 pH units (54%) in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.24 pH units above the initial resting pHj value, which reached its maximum ~ 5.4 min following the return to normoxia. The pH t response to anoxia induced by equilibrating HC0 37C0 2-buffered media with 95% Ar/5% C 0 2 for > 18 hours was not statistically different from the pHj response to anoxia induced with media containing 2 m M Na 2 S 2 0 4 (compare with Figure 9B). I l l Time (min) 112 Figure 12. Effects of the removal of extracellular Ca on the pH, response to a 5 min period of anoxia Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. A . Upon exposure to a Ca -free solution containing 200 u M EGTA, steady-state pH ; increased — 0.11 pH units and reached a plateau after ~ 7.2 min. pH ; remained stable during the following 25 min. B. Upon exposure to a Ca -free solution containing 200 p M EGTA, steady-state pHi increased. Once a new steady-state pH; of ~ 7.24 was reached, a 5 min period of anoxia induced an intracellular acidification that recovered slightly in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization that rose above the pre-anoxic steady-state pHj. The magrutude and the time course of the pH, response to a 5 min period of anoxia was not influenced by the presence or absence of extracellular Ca (compare with Fig. 7A, the same experiment performed in the presence of extracellular Ca 2 + ). Each trace represents data collected from a different neuron. 113 Time (min) 114 Figure 13. Effects of the removal of extracellular Ca on the Ca response to a 5 min period of anoxia The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. The resting BI334IBI3m ratio value (the ratio of the background-corrected I334 and 7,80) was ~ 0.45. Upon exposure to a Ca -free solution containing 200 p M EGTA, the BI334/BI3i0 ratio value decreased. During a 5 min period of anoxia, there was a small increase in the BI334/BI3S0 ratio value. Upon the return to normoxia, this cell displayed a further post- 2+ anoxic rise in the BI334/BI3M ratio value. Thus, the Ca response to a 5 min period of anoxia is dependent on the presence of extracellular Ca (compare with Fig. 10, the same experiment performed in the presence of extracellular Ca 2 +). 115 0 [Ca' ] o + 200 u M E G T A Anoxia I 1 1 1 1 1 1 1 1 0 5 10 15 20 25 30 35 40 Time (min) 116 Figure 14. Effects of exposure to reduced Na+, NMDG+-substituted medium on the pH; response to a 5 min period of anoxia Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. A . With a steady-state pHj of ~ 7.37, exposure to a 2 m M Na + , NMDG+-substituted medium caused a fall in pH,. During the continued perfusion with this reduced-Na+ medium, pHj gradually recovered and, over ~ 22 min, reached a new steady-state pH, value that approximated the pH; value observed prior to exposure to reduced-Na+ medium. B. With a steady-state pHj of ~ 7.22, exposure to a 2 m M Na + , NMDG+-substituted medium caused a fall in pH ; . pH; then recovered to ~ 7.40. A 5 min period of anoxia induced an intracellular acidification of ~ 0.10 pH units, the magnitude of which was smaller than observed in the presence of normal external Na + . pH, rose slightly in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.11 pH units, a magnitude which was smaller than observed in the presence of normal extracellular Na + (compare with Fig. 7A, the same experiment performed in the presence of normal extracellular Na + ; Table 6). Each trace represents data collected from a different neuron. . Time (min) 118 Figure 15. Effects of exposure to reduced Na+, Lf-substituted medium on the pHj response to a 5 min period of anoxia The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. Exposure to a 2 m M Na + , Li+-substituted medium caused pH ; to fall. Over - 1 2 min, pH ; recovered and reached a new steady-state pH ; value. A 5 min period of anoxia then induced an intracellular acidification that recovered slightly in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization. Under these conditions, the magnitudes of all three phases of the pHj response to anoxia were not different than those observed in the presence of normal external N a + (compare with Fig. 7A, the same experiment performed in the presence of normal external Na + ; Table 6). In contrast, the magnitudes of the per-anoxic acidification and per-anoxic alkalinization were significantly larger than those observed in the presence of reduced Na + , N M D G + - substituted media (compare with Fig. 14, the same experiment performed under reduced- Na+ NMDG+-substituted conditions; Table 6). 119 Time (min) 120 Figure 16. Effect of an anoxic period on the rate of pH, recovery from an intracellular acid load imposed during an anoxic period The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. A n initial acid load and subsequent recovery of pH; was conducted under control conditions. A second acid load was then performed during an anoxic period. The rate at which pHj recovered after the second intracellular acid load was decreased when compared to the control rate of pH; recovery.  122 Figure 17. Comparison of the rates of pH; recovery from intracellular acid loads imposed prior to and during an anoxic period Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. Plotted are the mean rates of pH, recoveries from intracellular acid loads conducted prior to (•) and during (O) an anoxic period, at pHj values denoted on the x-axis. The rates of pHj recovery were evaluated at 0.05 unit intervals of pHj 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 8 paired experiments of the type shown in Fig. 16. Except at pH ; 6.95, at every absolute level of pH i 5 the rates of pHj recovery were significantly slowed during an anoxic period as compared to the control rates of pHj recovery (p<0.05 at each absolute value of pH i 5 except at pHi 6.95). 0.020 0.018 J 124 Figure 18. Effect of a transient anoxic period on the rate of pH s recovery from an intracellular acid load The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. A n initial acid load and subsequent recovery of pH; was conducted under control conditions. A second acid load was then performed following a 5 min period of anoxia, at a time when steady-state pH, was increased. The rate at which pHj recovered after the second intracellular acid load was increased when compared to the control rate of pHj recovery.  126 Figure 19. Comparison of the rates of pH, recovery from intracellular acid loads imposed prior to and following a 5 min period of anoxia Experiments were performed under HCC^VCCVfree, HEPES-buffered conditions. Plotted are the mean rates of pH; recoveries from intracellular acid loads conducted prior to (•) and following (O) a 5 min period of anoxia, at pH; values 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 17 paired experiments of the type shown in Fig. 18. At each absolute level of pH i 5 the rates of pHj recovery were significantly faster following an anoxic period as compared to the control rates of pH; recovery (p<0.05 at each absolute value of pHj). 127 0.020 -, 128 Figure 20. Effects of 0.5 p M tetrodotoxin (TTX) on the steady-state pH, response to a 5 min period of anoxia The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions in the presence of 0.5 p M TTX. Upon exposure to a 5 min period of anoxia, the initial resting pH ; value decreased by ~ 0.07 pH units and recovered by ~ 0.03 pH units in the continued absence of oxygen. The absolute magnitudes of both of these phases were smaller than observed in the absence of TTX. Upon the return to normoxia, there was an intracellular alkalinization of ~ 0.21 pH units above the initial resting pH s value. The magnitude of the post-anoxic alkalinization was not statistically different than observed in the absence of T T X (compare with Fig. 7A, the same experiment performed in the absence of TTX; Table 7). Time (min) 130 Figure 21. Effect of 0.5 u M T T X on the rate of pH s recovery from intracellular acid loads imposed prior to and during the anoxic period The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions and 0.5 u M T T X was present throughout the course of the experiment. A n initial acid load and subsequent recovery of pH ( was conducted under normoxic conditions. A second acid load was then performed during an anoxic period at a time when steady-state pH ; was decreased. The rate at which pHj recovered after the second intracellular acid load was decreased when compared to the control rate of pH; recovery at the same absolute values of pH ( (compare with Fig. 16, the same experiment performed in the absence of TTX). Time (min) 132 Figure 22. Comparison of the rates of pH ( recovery from intracellular acid loads imposed prior to and during an anoxic period in the presence or absence of TTX Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. Plotted are the mean rates of pHj recoveries from intracellular acid loads conducted prior to (•) and during (O) an anoxic period in the presence of 0.5 p M TTX, at pHj values denoted on the x-axis. In addition, plotted are the mean rates of pH ; recoveries from intracellular acid loads conducted prior to (•) and during (•) an anoxic period in the absence of T T X (see Fig. 17). The rates of pH, 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 in the presence of TTX were obtained from 7 paired experiments of the type shown in Fig. 21. Both prior to and during an anoxic period, at each absolute level of pH i 5 the rates of pH ( recovery were not statistically different in the presence or absence of TTX. 133 134 Figure 23. Effect of 500 p M ZnCl 2 on the steady-state pH, response to a 10 min period of anoxia The experiment was performed under HC0 3VC0 2-free, HEPES-buffered conditions. The application of 500 p M ZnCl 2 did not influence steady-state pHj. A 10 min period of anoxia induced an intracellular acidification that was not significantly different than observed in the absence of ZnCl 2 . In the continued absence of oxygen, pHi recovered by - 0 . 0 4 pH units (29%) and, upon the return to normoxia, there was an intracellular alkalinization of - 0.13 pH units above the initial resting pHj value. The magnitudes of both the per-anoxic rise in pH f and the post-anoxic alkalinization were smaller in the presence of ZnCl 2 than observed in the absence of ZnCl 2 (compare with Fig. 8B, the same experiment performed in the absence of 500 p M ZnCl 2 ; Table 7). 135 136 Figure 24. Distributions of steady-state pH, values for all neurons during perfusion with HC037C02-free, HEPES-buffered and HC037C02-buffered media at 22°C A. The distribution of the resting pH ( values for 26 adult rat hippocampal CA1 neurons during perfusion with HC037C02-free, HEPES-buffered media at 22°C (bin width = 0.05 pH units). The data were best fitted by a Gaussian distribution with a mean at pHj 7.23 ± 0.02. B. The distribution of resting pHj values for 16 neurons during perfusion with HC037C02-buffered media at 22°C (bin width = 0.05 pH units) was fitted with a Gaussian distribution, around a mean of 7.24 ± 0.02.  138 Figure 25. Effects of a 5 min period of anoxia on steady-state pH; at 22°C A. Prior to an anoxic period in HC037C02-free, HEPES-buffered media at 22°C, the initial resting pH; value was - 7.15. A 5 min period of anoxia induced an intracellular acidification that did not show any recovery in the continued absence of oxygen. Upon the return to normoxia, there was a small intracellular alkalinization. The magnitudes of all three phases of the pHj response to anoxia were smaller at 22°C when compared to 37°C under identical buffering conditions (compare with Fig. 7A, the same experiment performed at 37°C; Table 8). B. Upon exposure to a 5 min period of anoxia in HC03" /C02-buffered media at 22°C, the initial resting pH; value of - 7.18 decreased. In the continued absence of oxygen, pH, recovered and, upon the return to normoxia, there was an intracellular alkalinization. Again, the magnitudes of all three phases of the pHt response to anoxia were smaller at 22°C when compared to 37°C, under identical buffering conditions (compare with Fig. 9B, the same experiment performed at 37°C; Table 8). Each trace represents data collected from a different neuron. 139 Time (min) 140 Figure 26. Effect of a transient period of anoxia on the rate of pH s recovery from an intracellular acid load at 22"C The experiment was performed under HC037C02-free, HEPES-buffered conditions. An initial acid load and subsequent recovery of pH; was conducted under control conditions at 22°C. A second acid load was then performed following a 5 min period of anoxia. The rate at which pHj recovered after the second intracellular acid load was increased when compared to the control rate of pHj recovery at 22°C. 141 Time (min) 142 Figure 27. Comparison of the rates of pHj recovery from intracellular acid loads imposed prior to and following 5 min periods of anoxia at 22°C and 37°C Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. Plotted are the mean rates of pH; recoveries from intracellular acid loads conducted prior to (•) and following (O) a 5 min period of anoxia at 22°C, at pH; values denoted on the x- axis. Also plotted are the mean rates of pHj recoveries from intracellular acid loads conducted prior (•) and following (•) a 5 min period of anoxia at 37°C, at pH s values denoted on the x-axis (also see Fig. 19). The rates of pH; recovery were evaluated at 0.05 unit intervals of pHj 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 collected at 22°C were obtained from 11 paired experiments of the type shown in Fig. 26. At 22°C, at each absolute level of pH i ; the rates of pH; recovery were significantly faster following an anoxic period compared to the control rates of pH ( recovery (p<0.05 at each absolute value of pHj). Both prior to and following a 5 min period of anoxia, at each absolute level of pH i 5 the rates of pH ; recovery were significantly faster at 37°C as compared to the corresponding rates of pHi recovery at 22°C (p<0.05 at each absolute value of pH (). * 143 144 Figure 28. Effects of reducing pH 0 to 6.60 on the pHj response to a 5 min period of anoxia The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. Upon exposure to a pH 6.60 solution, the initial steady-state pH ; declined and a new steady-state pH; of ~ 6.85 was reached. A 5 min period of anoxia induced an intracellular acidification which was not different from that observed under control conditions at pH 0 7.35. During the anoxic period, pHj recovered by ~ 0.03 pH units (21%), and, upon the return to normoxia, there was an intracellular alkalinization of ~ 0.02 pH units. The magnitudes of the per-anoxic rise in pHj and the post-anoxic alkalinization were smaller than observed under control conditions at pH 0 7.35 (compare with Fig. 7A, the same experiment performed at pH 0 7.35; Table 9). 145 Time (min) 146 Figure 29. Effect of a transient period of anoxia on the rate of pH; recovery from an intracellular acid load imposed at pH„ 6.60 The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. A n initial acid load and subsequent recovery of pH, was conducted prior to an anoxic period at pH 0 6.60. A second acid load was then performed following a 5 min period of anoxia at pH 0 6.60. The rate at which pHj recovered after the second intracellular acid load was increased when compared to the rates of pH; recovery observed prior to the induction of anoxia at pH 0 6.60. p H 0 6.60 Time (min) 148 Figure 30. Comparison of the rates of pH, recovery from intracellular acid loads imposed prior to and following a 5 min period of anoxia at pH 0 6.60 and pH„ 7.35 Experiments were performed under HC0 3VC0 2-free, HEPES-buffered conditions. Plotted are the mean rates of pHj recoveries from intracellular acid loads conducted prior to (•) and following (O) a 5 min period of anoxia at pH 0 6.60, at pHj values denoted on the x-axis. Also plotted are the mean rates of pH ( recoveries from intracellular acid loads conducted prior to (•) and following (•) a 5 min period of anoxia at pH 0 7.35 (also see Fig. 19). The rates of pHj recovery were evaluated at 0.05 unit intervals of pH t 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 collected at pH„ 6.60 were obtained from 9 paired experiments of the type shown in Fig. 29. At pH 0 6.60, at each absolute level of pHi except pH, 6.70, the rates of pH s recovery were significantly faster following a 5 min period of anoxia as compared to the control rates of pHj recovery at pH 0 6.60 (p<0.05 at each absolute value of pH ; , except at pH ; 6.70). Both prior to and following a 5 min period of anoxia, at each absolute level of pH i 5 the rates of pH; recovery were significantly faster at pH 0 7.35 as compared to the corresponding rates of pH; recovery observed at pH 0 6.60 (p<0.05 at each absolute value of pH;). 149 150 Figure 31. Effects of increasing pH„ to 7.60 on the pHj response to a 5 min period of anoxia Upon exposure to a HC0 37C0 2-free, HEPES-buffered solution, pH 7.60, the initial steady-state pHj increased to reach a new steady-state pHj of ~ 7.92. A 5 min period of anoxia caused an intracellular acidification that was smaller than observed at pH 0 7.35. In the continued absence of oxygen, pHj recovered and, upon the return to normoxia, there was a large intracellular alkalinization. The magnitudes of the per-anoxic rise in pHj and the post-anoxic alkalinization were larger than observed under control conditions at pH 0 7.35 (compare with Fig. 7A, the same experiment performed at pH 0 7.35; Table 9). Time (min) 152 Figure 32. Effect of a transient anoxic period on the rate of pH ; recovery from an intracellular acid load at pH„ 7.60 The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. A n initial acid load and subsequent recovery of pHj was conducted prior to an anoxic period at pH 0 7.60. A second acid load was then performed following a 5 min period of anoxia at pH 0 7.60. The rate at which pH ; recovered after the second intracellular acid load was increased when compared to the control rate of pHj recovery at pH„ 7.60. Time (min) 154 Figure 33. Comparison of the rates of pHs recovery from intracellular acid loads imposed prior to and following a 5 min period of anoxia at pH„ 7.60 and pH 0 7.35 Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. Plotted are the mean rates of pH, recoveries from intracellular acid loads conducted prior to (•) and following (O) a 5 min period of anoxia at pH 0 7.60, at pH ; values denoted on the x-axis. Also plotted are the mean rates of pHj recoveries from intracellular acid loads conducted prior to (•) and following (•) a 5 min period of anoxia at pH 0 7.35, at pHj values denoted on the x-axis (see also Fig. 19). The rates of pHj recovery were evaluated at 0.05 unit intervals of pHj 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 collected at pH 0 7.60 were obtained from 7 paired experiments of the type shown in Fig. 32. At pH 0 7.60, the rates of pHj recovery were significantly faster following a 5 min period of anoxia compared to the corresponding control rates of pHj recovery at pH 0 7.60 (p<0.05 at each absolute value of pH ; except at pH, 7.20). Both prior to and following a 5 min period of anoxia, at every absolute level of p H b the rates of pH; recovery were significantly faster at pH 0 7.60 when compared to the corresponding rates of pHi recovery observed at pH 0 7.35 (p<0.05 at each absolute value of pHj). 155 156 Figure 34. Relationship between pH 0 and pH, in HC037C02-free, HEPES-buffered media under normoxic conditions Plotted are the pH values of the superfusing media (pH0) against the resulting mean steady-state pH; values. Error bars represent S.E.M. The continuous line represents the least squares linear regression fit to the data points. The numbers in parentheses above or below the data point indicate the number of individual neurons from which the observation was made. The slope of the linear regression line is ~ 0.74.  158 Figure 35. Effects of 3, 5 and 10 min periods of anoxia on steady-state pH, during perfusion with media containing 5.0 mM glucose Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. A . Upon the induction of a 3 min period of anoxia, there was an intracellular acidification. pHi did not recover in the continued absence of oxygen. The magnitude of the per-anoxic rise in pH ; was smaller than observed in the presence of 17.5 m M (compare with Fig. 8A, the same experiment performed in the presence of 17.5 m M glucose; Table 10). Upon the return to normoxia, there was an intracellular alkalinization above the initial resting pH; value which was not different than observed in the presence of 17.5 m M glucose. B. Upon the induction of a 5 min period of anoxia, the initial resting pHj value of ~ 7.26 decreased and, in the continued absence of oxygen, recovered slightly. The magnitudes of the per-anoxic acidification and per-anoxic rise in pH; were smaller than observed in the presence of 17.5 m M glucose (compare with Fig. 7A, the same experiment performed in the presence of 17.5 m M glucose; Table 10). Upon the return to normoxia, there was an intracellular alkalinization that was not different than observed in the presence of 17.5 m M glucose. C. Upon exposure to a 10 min period of anoxia, there was an intracellular acidification, the magnitude of which was smaller than observed in the presence of 17.5 m M glucose (compare with Fig. 8B, the same experiment performed in the presence of 17.5 m M glucose; Table 10). In the continued absence of oxygen, pHj recovered, and upon the return to normoxia, there was an intracellular alkalinization, neither of which were different than observed under identical buffering conditions in the presence of 17.5 m M glucose. Each trace represents data collected from a different neuron. 159 Time (min) 160 Figure 36. Effects of exposure to reduced Na* medium on the pH : response to a 5 min period of anoxia in media containing 5.0 mM glucose Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. A. Upon exposure to a 2 m M Na + , N M D G + substituted medium, pH ; fell from an initial steady-state pHj of ~ 7.44. Over - 1 2 min, pH ; recovered and reached a new steady-state pH; value. A 5 min period of anoxia caused an intracellular acidification that recovered slightly in the continued absence of oxygen. Upon the return to normoxia, there was an intracellular alkalinization that was smaller than observed in the presence of normal extracellular N a + and 5.0 m M glucose (compare with Fig. 35B, the same experiment performed in the presence of normal extracellular Na + ; Table 11). B. Upon exposure to 2 m M Na + , L i + substituted media, pH ; decreased and gradually recovered to a new steady- state pHj value of ~ 7.22. A 5 min period of anoxia induced an intracellular acidification that recovered slightly in the continued absence of oxygen. Upon the return to normoxia, pHj increased ~ 0.22 pH units above the initial steady-state pHj. The magnitude of the post-anoxic alkalinization was not different than observed in the presence of normal extracellular Na + (compare with Fig. 35B, the same experiment performed in the presence of normal extracellular Na + ; Table 11). Each trace represents data collected obtained from a different neuron. Time (min) Time (min) 162 Figure 37. Effects of 50 uM Rp-cAMPS and 10 pM Noradrenaline on the pH, response to a 5 min period of anoxia Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions. In the absence of any pharmacological treatments (•), a 5 min period of anoxia induced a fall in pHj of ~ 0.12 pH units that recovered by ~ 0.05 pH units (42%) in the continued absence of oxygen. Upon the return to normoxia, pH, increased -0.16 pH units above the initial steady-state pH;. In the presence of 50 p M Rp-cAMPS (an inhibitor of P K A ; A ) , the magnitude of all three phases of the pHj response to a 5 min period of anoxia were reduced (Table 12). In the presence of 10 p M noradrenaline (an activator of P K A ; • ) , the magnitude of the post-anoxic alkalinization was larger than observed under control conditions (Table 12). Each trace represents data collected from a different neuron. 163 164 DISCUSSION The results presented in this thesis are the first detailed description of the effects of anoxia on intracellular pH in adult mammalian central neurons, under conditions where the pH; response to anoxia can be attributed to mechanisms intrinsic to the neurons under study. Although previous studies have documented the effects of anoxia on pHj both in vivo and in slice preparations in vitro (e.g. Whittingham et al, 1984; Silver and Erecinska, 1990; Eleff et al, 1991; Fujiwara et al, 1992; Vornov et al, 1996), it is difficult (if not impossible) to separate the contribution of various cell types (including glia) to the effects observed under these experimental conditions. In addition to characterizing anoxia-evoked changes in steady-state pHi, the mechanisms underlying these changes were examined, with particular reference to the role of changes in the activity of the NaVFf exchanger. Not only is the latter transport mechanism the dominant acid extrusion pathway in rat hippocampal neurons (Raley-Susman et al, 1991; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998), but also changes in NaVFf exchange activity have been shown to be critically important for determining cellular viability in both peripheral cell types and in central neurons following ischemia-like insults (Bond et al, 1993; Harper et al, 1993; Vornov et al, 1996). Finally, results presented in the thesis also contribute to our understanding of the control of pHi in hippocampal neurons under normoxic conditions. 165 1 Intracellular pH Regulation Under Normoxic Conditions 1.1 Intracellular pH regulating mechanisms present in rat hippocampal CA1 neurons Under physiological conditions, three mechanisms have been found, to date, to contribute to pH; regulation in rat hippocampal neurons; an acid-extruding Na7H + exchanger, an acid-extruding Na+-dependent HC03"/C1" exchanger, and an acid-loading Na+-independent HC037C1" exchanger (Raley-Susman et al, 1991 and 1993; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al, 1996; Smith et al, 1998). Although it has been suggested that the Na+-dependent HC0 3 7Cr exchanger is the most important acid extrusion mechanism present in this cell type (Schwiening and Boron, 1994), this laboratory (Baxter and Church, 1996; Smith et al, 1998) and the laboratories of others (Raley-Susman et al, 1991; Bevensee et al, 1996) have provided evidence that Na7H + exchange is, in fact, the dominant acid extrusion mechanism. In support, in the present study, the mean steady-state pH t was not influenced by the presence or absence of extracellular HC0 3 " . Evidence has been found in the present study for the existence of additional mechanism(s) which contribute to pH s regulation in rat hippocampal neurons. Thus, pH; regulation was observed even in the absence of extracellular N a + under HC0 37C0 2-free, HEPES-buffered conditions, suggesting the presence of a Na + - and HC03"-independent pHj regulatory mechanism(s) in this cell type (also see Bevensee et al, 1996). This is perhaps not surprising, given the very large number of Na + - and HC03"-independent mechanisms which have been found to participate in pHj regulation in a variety of 166 peripheral cell types (e.g. Kurtz, 1987; Blatter and McGuigan, 1990; Gibb et al, 1997; Karwatowska-Prokopczuk et al, 1998). These include, for example, voltage-dependent proton conductances (gu+; Byerly and Suen, 1989; Kapus et al, 1993; Gordienko et al, 1996), lactate/Ff cotransport (Poole and Halestrap, 1993; Gibb et al, 1997), Ca 2 7Ff ATPase (Schwiening et al, 1993), V - and P-type H + ATPases (Nelson and Klionsky, 1996; Karwatowska-Prokopczuk et al, 1998), and K 7 H + exchange (Sachs et al, 1976; Hofer and Machen, 1992; Wilding et al, 1992; Hogan et al, 1995a and 1995b). Preliminary evidence is presented in this thesis that Ca 2 7H + ATPase, lactate/FT cotransport, gH+, and/or K 7 H + exchange activity may exist in hippocampal neurons, given the fact that maneuvers which influence the activities of each of these mechanisms are able to affect the recovery of pH, during exposure to Na+-free, NMDG+-substituted media. However, additional experiments are required to assess formally the potential roles of these mechanisms in pH ; regulation in rat hippocampal neurons. The possibility remains that these mechanisms may be dormant under physiological conditions and be activated under particular (i.e. [Na+]0-free) conditions (see Kurtz, 1987; Peral et al, 1995). 1.2 The role of extracellular pH in determining intracellular pH In contrast to many peripheral cell types in which pHj is not markedly affected by changes in pH„ (e.g. Aickin, 1984; Wilding et al, 1992), pH ; in mammalian central neurons appears to be steeply dependent upon pH 0 . Thus, the ratio ApH^ApH,, (the slope of the linear regression line relating pHj to pH0) is approximately 0.7 in rat brain synaptosomes (Sanchez-Armass et al, 1994), cultured fetal rat neocortical neurons (Ou- 167 Yang et al, 1993) and cultured fetal rat hippocampal neurons (Church et al, 1998). The present study confirms that the steep dependency of pH, on pH 0 also exists in adult rat hippocampal neurons, a finding which suggests that changes in pH; may account for some of the known effects of changes in pH 0 on hippocampal neuron function (see; Church and McLennan, 1989; Church, 1992; Tombaugh and Somjen, 1996 and 1997; Balestrino and Somjen, 1998; Church et al, 1998). In both peripheral cell types (Vaughan-Jones and Wu, 1990; Peral et al, 1995) and in rat brain synaptosomes (Jean et al, 1985), NaVFf exchange activity is influenced by changes in pH D and thereby provides a mechanism to link changes in pH 0 to changes in pHj. While Jean et al (1985) suggested that, in rat brain synaptosomes, the transmembrane proton gradient determines the rate of activity of the exchanger, Vaughan-Jones and Wu (1990), using cardiac Purkinje fibers, found that the absolute magnitudes of both pH 0 and p l f determine NaVFf activity. In both cases, however, an increase in pH 0 shifted the pH; dependence of the exchanger in the alkaline direction, thereby increasing the activity of the exchanger. Conversely, a decrease in pH 0 caused a decrease in pH rmediated stimulation of acid extrusion (Jean et al, 1985; Vaughan-Jones and Wu, 1990). In the present study, changes in pH 0 affected the activity of the NaVFf exchanger as judged by changes in the slopes of the regression lines relating pH; to the rates of change of pHi following internal acid loads imposed under HC03"-free conditions. Thus, NaVFf exchange activity in adult rat hippocampal neurons is inhibited by a reduction in pH 0 whereas its activity is stimulated with an increase in pH 0 . In addition to influencing 168 the rate of Na7H + exchange activity, changes in pH 0 evoked shifts in the pH; dependence of Na7H + exchange, as there was a progressive alkaline shift in the x-intercept values of the linear regression lines relating pHj to rates of pH, recovery from imposed intracellular acid loads under pre-anoxic conditions as pH 0 was increased. Thus, at pH 0 6.60, the increased concentration of extracellular protons appears not only to directly inhibit Na7H + exchange but also decrease the intracellular affinity of the exchange mechanism for protons, thus further reducing the rate of acid extrusion. Conversely, at pH 0 7.60, there was a rapid rate of acid extrusion. Therefore, in rat hippocampal neurons, pH 0 appears to be able to influence pHj by influencing the activity of the Na7H + exchanger. The relevance of these findings to the neuronal response to anoxia is discussed in Section 6.3.1. 1.3 The role of ambient temperature in determining intracellular pH It is well known that steady-state pH varies with temperature; in H C 0 3 7 C 0 2 - buffered media, pH rises as temperature rises whereas, for HC0 37C0 2-free media, pH falls as temperature rises. In the present experiments, under conditions where pH 0 was maintained at a constant value (pH 7.35), as temperature was reduced from 37°C to 22°C, steady-state pIL declined both in the presence and absence of H C 0 3 7 C 0 2 . This result contrasts with those of Marjanvoic et al (1998) and Laptook et al (1995a and 1995b) who observed an inverse relationship between brain temperature and cerebral pH ; in vivo. The results of the present study suggest that the reduced steady-state pH, values observed in isolated adult hippocampal neurons at 22°C may, at least in part, reflect reduced Na7H + 169 exchange activity, as evidenced by the reduced slope of the relationship between pHj vs. rate of pHj recovery from imposed acid loads as temperature is reduced. A similar, steep dependence of Na7H + exchange activity on ambient temperature has previously been demonstrated for NHE1, NHE3, and for the N H E isoform present in hippocampal neurons (Baxter and Church, 1996; Bookstein et al, 1997) and has been suggested to reflect a change in the relative protonation of a-imidazole sites on histidine residues present on the Na7H + exchanger (Hauge and Kofstad, 1995). The relevance of the temperature-dependence of Na7H + exchange activity to anoxia is discussed in Section 6.3.2. 2 The Intracellular pH Response to a Transient Anoxic Period pH, transients are observed under both physiological and pathophysiological conditions and play a role in regulating neuronal function (see Introduction, Section 3.3). In acutely dissociated adult rat hippocampal CA1 neurons, a transient anoxic period induced an intracellular acidification that recovered slightly in the continued absence of oxygen, followed by a post-anoxic internal alkalinization. A similar pattern of an anoxic acidification followed by a post-anoxic alkalinization has been observed in cortical brain slices in vitro and in rat whole brain following ischemia, although, as noted above, the contribution of neuronal elements to the pH; changes observed in these preparations could not be assessed (Mabe et al, 1983; Pirttila and Kauppinen, 1992). The pH; response to a transient period of anoxia observed in the present study is also similar, in many respects, 170 to the neuronal pH; changes induced by seizure activity or the application of exogenous glutamate (Siesjo et al, 1985; Hartley and Dubinsky, 1993; Ou-Yang et al, 1995). 2.1 Studies of the intracellular pH response to anoxia in vivo and in slice preparations in vitro The majority of studies have investigated the pH; response to anoxia/ischemia using in vitro slice preparations and have reported an intracellular acidification that, upon reoxygenation, recovers to pre-anoxic steady-state pHj levels; however, wdth the exceptions noted above, per-anoxic rises in pHj and marked post-anoxic internal alkalinizations have not been commonly observed (Whittingham et al, 1984; Eleff et al, 1991; Fujiwara et al, 1992). A similar pattern of pH; changes has also been observed when measuring the pH; changes of CA1 neurons in response to a transient period of ischemia in vivo (Silver and Erecinska, 1992). The pHj responses observed in the in vitro preparations are a composite average of pH; changes occurring in numerous cell types which may respond differentially to anoxia/ischemia. Indeed, this has been suggested to underlie the heterogeneity of cellular pHj changes observed in response to H C 0 3 7 C 0 2 removal in cortical brain slices (Pirttila and Kauppinen, 1994b). Moreover, Kraig et al (1986) suggested that during anoxia, protons are heterogeneously distributed between separate neuronal and glial compartments, each contributing to the overall pHj response to ischemia/anoxia (but see Katsura et al, 1992a). In addition, in in vivo studies and in studies using brain slices, extracellular pH (pH0), a variable that directly influences pHj and the activities of pH ; regulating mechanisms (see Section 1.2), is not controlled, and, 171 in fact in the latter preparation, varies along the depth of the slice (Krnjevic and Walz, 1990). Each of these potentially confounding variables are minimized when utilizing acutely dissociated hippocampal neurons and, thus, the results of this study illustrate the inherent neuronal pHj response to a transient anoxic insult. If pH 0 is reduced during and immediately following an anoxic insult, as occurs in vivo, the pH ; response observed in acutely dissociated neurons resembles the pHj response expected to occur both in slice preparations and in vivo (see Section 6.3.1). This observation strongly supports both the validity of the neuronal pH; response to anoxia observed in the present study as well as the method used to induce anoxia. 2.2 The role of extracellular HC03~ in the production of the intracellular pH response to anoxia While an extensive literature exists concerning changes in the activities of NaTFf exchangers during and following a transient period of anoxia (see Sections 4.3 and 6.2), the potential role of HC03'-dependent pH ; regulatory mechanisms in the pH> response to anoxia remains largely unknown. HC03'-dependent pH; regulating mechanisms have been shown to be involved in the regulation of pH 0 during spreading depression and cerebral ischemia (Mutch and Hansen, 1984), and have been shown to contribute to the recovery of pH ( following ischemia in cardiac tissue (Vandenberg et al, 1993) and following glutamate excitotoxicity in cultured hippocampal neurons (Hartley and Dubinsky, 1993). Conversely, in cortical brain slices, DIDS fails to influence the pH; response to hypoxia (Pirttila and Kauppinen, 1994a). In further support for the lack of 172 involvement of changes in the activities of HC03"-dependent pH; regulating mechanisms, in the present study the pHj response to anoxic periods of various durations was statistically similar in all respects in the presence or absence of extracellular HC0 3 ". Thus, HC03"-dependent pHj regulating mechanisms are unlikely to be major contributors to the pH ; response to anoxia in adult rat hippocampal CA1 neurons, suggesting that the Na7H + exchanger may be central to the production of the pHj response to transient periods of anoxia in this cell type. Despite the present results which suggest that changes in the activities of HC0 3 " - dependent pH ( regulatory mechanisms are not involved in the production of the pHj response to transient periods of anoxia, their possible contribution cannot be unequivocally ruled out. The presence of extracellular HC0 3 " has been shown to both stimulate Na7H + exchange in renal epithelia (Chen and Boron, 1995) and inhibit Na7H + exchange in a mouse embryo cell line (Gillies and Martinez-Zaguilan, 1991). Furthermore, the presence of extracellular H C 0 3 " increases extracellular buffering capacity, an effect which, independent of HC03"-dependent transport mechanisms, inhibits the magnitude of the pH; alkalinization observed following an hypoxic insult in cortical neurons (Pirttila and Kauppinen, 1993). 3 The Intracellular Calcium Response to a Transient Anoxic Period 2+ A profound and rapid increase in intracellular [Ca ] is consistently observed during periods of anoxia/ischemia (Lipton and Lobner, 1990; Mitani et al, 1994), excitotoxicity (Wang et al, 1994; Budd and Nicholls, 1996), and spreading depression 173 (Erecinska and Silver, 1996). In fact, the size and duration of these internal C a 2 + transients, as well as their origin, may directly determine subsequent neuronal survival (Tymianski et al, 1993; Kristiane and Siesjo, 1997). Among the most sensitive regions of the brain to anoxia-induced neuronal damage is the hippocampal C A I region and, in the present study, there was a rapid increase in [Ca2 +] ; within two minutes of the initiation of an anoxic insult and [Ca \ remained elevated following the return to normoxia. These data confirm those of Friedman and Haddad (1993), whose laboratory used an identical tissue preparation and method of inducing anoxia. Considering the dispersed nature of these dissociated cells, along with the constant flow of perfusate, an anoxia-evoked release of glutamate and subsequent activation of Af-methyl-D-aspartate receptors appears unlikely to be able to account for the 2+ rapid influx of extracellular Ca , although this possibility was not directly examined. 2+ Rather, the initial increase in [Ca \ is likely due to the activation of voltage-sensitive calcium channels consequent upon membrane depolarization and the subsequent influx of extracellular Ca 2 + (also see Friedman and Haddad, 1993). Interestingly, [Ca2+]j did not recover even upon the return to normoxia (also see Friedman and Haddad, 1993), suggesting that the neurons are unable to repolarize in the immediate post-anoxic period (Tanaka et al, 1997; Yamamoto et al, 1997). The combination of membrane depolarization and an anoxia-evoked elevation in [Na+]j (see Section 4.3) may lead to the reversal of the Na7Ca 2 + exchanger which could also contribute to maintaining an elevated [ C a 2 ! (Stys, 1998). 174 In the absence of extracellular Ca 2 + , a small and transient increase in [Ca2 +]j occurred in the post-anoxic period which may tentatively be attributed to the release of C a 2 + from intracellular stores. Inositol 1,4,5-trisphosphate- (InsP3) -sensitive, rather than 2+ Ca -sensitive, stores have been described in hippocampal neurons and have been shown to be involved in the anoxic response of these cells (Belousov et al, 1995; Erecinska and 2+ Silver, 1996). Thus, intracellular and extracellular Ca both likely contribute to the production of the anoxia-evoked increase in [Ca2+]j in acutely dissociated adult rat CA1 hippocampal neurons. 3.1 Role of calcium in the production of the intracellular pH response to anoxia 2+ A n increase in [Ca ]j can induce an intracellular acidification at least in part by competing with protons for shared intracellular binding sites (see Ives and Daniel, 1987). Influx of C a 2 + can also stimulate Ca 2 7H + ATPase activity both in snail neurons (e.g. Schwiening et al, 1993) and, possibly, in rat hippocampal neurons (Trapp et al, 1996), an effect which would also promote an internal acidification. However, in the present study, neither the fall in pHj evoked by anoxia (nor any of the other phases of the pHj response to anoxia) were influenced by the removal of extracellular Ca 2 + . In addition, the time course of the pHj response to anoxia was not influenced by the removal of extracellular Ca 2 + . Thus, anoxia-evoked changes in pHj are not secondary to anoxia-evoked changes in 2+ [Ca ]j in acutely dissociated adult rat hippocampal CA1 neurons. 175 4 Factors Contributing to the Anoxic Acidification A decrease in pHj is often the first observed event in the neuronal response to an anoxic or ischemic insult and, as such, may initiate and/or regulate subsequent changes in neuronal function (Silver and Erecinska, 1990; Martin et al, 1994). In the present study, anoxic periods of either 3, 5 or 10 min duration consistently produced an initial intracellular acidification. Previous studies have attributed the intracellular acidification to either the failure to regenerate ATP or to the increased production of metabolic acids (e.g. lactic acid). In addition, the present study suggests that a reduction in NaVFf exchange activity during anoxia may also contribute to the fall in pH t observed. 4.1 Intracellular A TP during anoxia Within the central nervous system, under normoxic conditions, ATP is primarily generated through oxidative phosphorylation; however, this process is inhibited during anoxia and glycolysis becomes the sole source of ATP. Unlike oxidative phosphorylation, glycolysis does not recycle the protons that are liberated during A T P hydrolysis (Dennis et al, 1991; Katsura et al, 1992b). Thus, even in the absence of lactate accumulation (see Section 4.2), protons accumulate due to an increasing dependence on anaerobic metabolism (Smith et al, 1993; Espanol et al, 1998). Given the profound reduction in intracellular ATP observed during anoxia (Lipton and Lobner, 1990; Mitani et al, 1994), the release of protons during ATP hydrolysis likely contributes to the intracellular acidosis evoked by anoxia in the present study. 176 The Na7K + ATPase is the primary consumer of ATP in neurons (Erecinska and Silver, 1989); thus, ATP levels may be preserved during periods of anoxia by maintaining the transmembrane N a + gradient, thereby reducing Na7K + ATPase activity. Indeed, in rat hippocampal slices, inhibition of N a + influx during anoxia attenuates anoxia-evoked falls in ATP (Hansen, 1985; Boening et al, 1989; Fried et al, 1995; Fowler and L i , 1998). Given that ATP hydrolysis contributes to the intracellular acidification observed during anoxia (see above), it follows that either the application of T T X or the removal of extracellular Na + should attenuate the magnitude of the anoxic acidification. Indeed, this is what was observed in the present study. Alternatively, it has been suggested that T T X may inhibit the anoxic acidification by reducing the anoxia-evoked Ca influx mediated by reverse Na7Ca 2 + exchange (Yamaguchi et al, 1997). However, the present study does not support this possibility in that neither the magnitude of the acidification, nor its time 2"*" course, was affected by the removal of extracellular Ca . 4.2 Intracellular lactate during anoxia The end-products of anaerobic glycolysis are two molecules of lactic acid per molecule of glucose metabolized. It has been suggested that anoxia-evoked reductions in pHj are directly proportional to intracellular lactate accumulation, which is, in turn, determined by the pre-anoxic/ischemic glucose concentration (Ljunggren et al, 1974; Smith et al, 1986; White et al, 1989; Katsura et al, 1992a; Roberts Jr. et al, 1998). However, others have suggested that a discontinuous relationship exists between internal lactate concentrations and pHj (Kraig et al, 1986). In support of a continuous relationship 177 between lactate accumulation and pHj, in the present study a significantly smaller intracellular acidification was evoked by anoxia in the presence of 5.0 m M glucose compared with 17.5 m M glucose. Indeed, the absolute magnitudes of the anoxia-evoked intracellular acidifications produced in the present study in the presence of 5.0 m M or 17.5 m M glucose are similar to those reported in rat hippocampal slices when the pre- anoxic glucose concentration was varied from 5 -15 m M (Roberts Jr. et al, 1998). The ability of lactate to produce an intracellular acidification is reduced under reduced-Na+ conditions (Pirttila and Kauppinen, 1992), a finding which may contribute to the attenuation of the anoxic acidification observed under reduced-Na+ conditions in the presence of 17.5 m M glucose. The absence of a similar effect in the presence of 5.0 m M glucose may be secondary to the reduced accumulation of lactate. 4.3 Changes in Na+/FT exchange activity In cardiac tissue, NaVFf exchange inhibition does not influence the magnitude of the intracellular acidification observed in response to ischemia (Askenasy et al, 1996; Piper et al, 1996). These results suggest that either the exchanger is not involved in pH; regulation during anoxia (Pike et al, 1993; Askenasy et al, 1996) or that the exchanger is inhibited during anoxia (Piper et al, 1996; Maddaford and Pierce, 1997). In support of the latter possibility, the present data indicate for the first time that, in rat hippocampal neurons, the NaVFf exchanger is inhibited during an anoxic period and may contribute to the production of the observed intracellular acidification. Thus, the activity of the NaVFf exchanger was significantly slowed under anoxic conditions and there was an acidic shift 178 in its pHj sensitivity. A similar inhibition of Na7H + exchange activity has been demonstrated during perfusion with the metabolic inhibitor, 2-deoxyglucose, in cardiac myocytes (Wu and Vaughan-Jones, 1994). However, despite its reduced activity, the Na7H + exchanger in hippocampal neurons is still partially active during anoxia. As has been demonstrated in peripheral cell types (Pike et al, 1993; Satoh et al, 1995; but see Tani and Neely, 1989; RuP et al, 1996), this residual activity may contribute to anoxia- evoked increases in [Na+]; which occur in acutely dissociated adult rat hippocampal C A I neurons (Fig 38; also see Friedman and Haddad, 1994b). In addition, this residual Na7H + exchange activity may contribute to the reduction in pH 0 seen during anoxia in vivo, as well as the production of anoxia-induced cellular swelling (Jakubovicz and Klip, 1989; Obrenovich et al, 1990). Thus, the present data indicate that the Na7H + exchanger present in rat hippocampal neurons is inhibited during an anoxic period. What is the identity of the factor(s) causing the reduction in Na7H + exchange activity? Firstly, during an anoxic period, increased phosphocreatine (PCr) and ATP hydrolysis produce a rise in the intracellular concentrations of inorganic phosphates, which are important physiological intracellular buffers (Burton, 1978; Wu and Vaughan-Jones, 1994). A n increase in PJ could potentially contribute to the observed decrease in the rate of acid extrusion. Arguing against this possibility, however, are the present findings that p( does not change dramatically during an anoxic period. Secondly, in peripheral cell types in vivo, anoxia- induced inhibition of Na7H + exchange is a result of a decrease in pH 0 (see Section 1.2); 179 however, in the present study, pH 0 was maintained at 7.35 throughout the anoxic period and yet a reduction in Na7H + exchange activity was still observed. An alternate explanation for the inhibition of Na7H + exchange activity observed during anoxia in the present study is that the activity of the Na7H + exchanger in rat hippocampal neurons may be dependent upon intracellular ATP levels. In rat hippocampal slices, there is an ~ 50% reduction in intracellular ATP levels after 5 min of anoxia (Boening et al, 1989; Lipton and Lobner, 1990). For Na7H + exchange isoforms 1, 2 and 3, ATP depletion causes both a decrease in the V m a x and a reduced sensitivity of the antiporters for internal protons (Cassel et al, 1986; Little et al, 1988; Levine et al, 1993; Kapus et al, 1994). These changes in Na7H + exchange activity are analogous to those changes observed in the present study during an anoxic period. ATP depletion inhibits Na7H + exchange activity independent of changes in the phosphorylation levels of the exchangers and in the presence of maintained ionic gradients (Goss et al, 1994). In a manner similar to pVadrenoceptor stimulation of Na7H + exchange activity (Hall et al, 1998), an associated regulatory protein may sense intracellular ATP levels and, in turn, influence Na7H + exchange activity (Goss et al, 1994). Although the internal ATP- dependence of the Na7H + exchanger present in rat hippocampal neurons has not been formally assessed, the possibility remains that the reduction in Na7H + exchange activity observed during anoxia in the present study may reflect anoxia-evoked reductions in intracellular ATP concentration. Nevertheless, it should be noted that T T X would be expected to preserve ATP levels during anoxia (see Section 4.1), however, Na7H + exchange activity during anoxia was reduced to a similar extent in the presence or 180 absence of TTX. This incongruity may imply that: i) TTX maintains internal ATP at a level such that the activities of secondary active transport mechanisms (i.e. Na7H + exchange) are reduced while, given its preferential sensitivity for ATP derived from glycolysis as well as its low for ATP, the Na7K + ATPase is functional, albeit at a slower rate (Erecinska and Silver, 1989); or ii) alternate mechanisms, e.g. anoxia-evoked changes in second messenger systems, are acting to inhibit Na7H + exchange activity during anoxia. As opposed to the reduced levels of intracellular ATP inhibiting Na7H + exchange activity during an anoxic period, the rapid restoration of intracellular ATP levels following an anoxic period may contribute to the post-anoxic activation of Na7H + exchange (see Section 6.2). Indeed, in cardiac myocytes, the application of exogenous ATP produces an intracellular alkalinization as a result of enhancing Na7H + exchange activity and shifting the sensitivity of the antiporter to more alkaline values (Puceat and Vassort, 1995). While the roles of ATP hydrolysis and lactate accumulation are further supported by the present data, this study suggests that inhibition of Na7H + exchange may also play a role in the production of the intracellular acidification seen during a transient anoxic insult. Residual Na + /H + exchange activity may be linked to the anoxia-evoked increase in [Na+]j and decrease in pH 0 evoked by anoxia in vivo. 181 4.4 Physiological relevance of the anoxic acidification The physiological importance of the intracellular acidification seen during an anoxic insult is confused by conflicting data which indicate that, on the one hand, acidosis is neuroprotective and, on the other hand, that it is detrimental to neuronal survival (see Introduction, Section 3.3). However, the relationship between a decrease in pHi and neuronal viability may be governed by a step function (Tombaugh and Sapolsky, 1993; L i and Siesjo, 1997). Thus, on the one hand, mild intracellular acidosis improves the functional recovery of hippocampal slices following anoxia (Roberts Jr. et al, 1998) and inhibits anoxia-induced neuronal damage (Giffard et al, 1990), possibly by stimulating glycolysis and/or reducing anoxia-induced intracellular C a 2 + overload (Tombaugh and Somjen, 1997). On the other hand, excessive and/or prolonged acidosis is detrimental to neuronal survival following anoxia (Nedergaard et al, 1991). For example, a large intracellular acidification of approximately 1.0 pH units increases the size of the infarcted core following ischemia/anoxia (Kraig et al, 1987). In the present study, the reduced intracellular acidification observed in response to an hypoxic/anoxic insult in the presence of T T X or mild hypothermia may reflect the increased likelihood of cellular recovery under these conditions. Although conflicting results have been reported under in vivo and in vitro preparations (Li and Siesjo, 1997), a similar step function may also explain the ability of variations in pre-ischemic extracellular glucose concentrations and lactate accumulation during an anoxic period to influence neuronal viability following an anoxic period. Thus, while increasing glucose concentrations slightly, from 5 to 15 mM, improves the 182 recovery of synaptic function following anoxia (Roberts Jr., et al, 1998), a significant increase in pre-ischemic glucose concentration, to ~ 25 mM, inhibits the recovery of ATP and PCr levels in the post-anoxic period and augments neuronal damage (LeBlanc et al, 1993; Tyson et al, 1993; L i and Siesjo, 1997). Similarly, while intracellular lactate has been suggested to be the preferred neuronal energy source in the post-anoxic period (Schurr et al, 1988; Schurr et al, 1997), a number of studies have documented the neurotoxic effects of marked lactic acidosis (Kraig et al, 1997; Siesjo et al, 1996). 5 Factors Contributing to the Per-Anoxic Rise in Intracellular pH In the continued absence of oxygen, pH ; slowly recovered from the maximum intracellular acidification induced by anoxia at a rate of ~ 0.04 pH units min'1. A similar per-anoxic recovery of pH; has previously been observed during a transient period of glucose and oxygen deprivation in mouse hippocampal slices (Fuijwara et al, 1992; Melzian et al, 1996). 5.1 Role of a voltage-activated proton conductance In response to anoxia/ischemia, hippocampal neurons transiently hyperpolarize. This hyperpolarization is quickly followed by a slow depolarization, which concludes in a rapid depolarization (Tanaka et al, 1997; Yamamoto et al, 1997). This rapid anoxic depolarization occurs wtithin the first few minutes following the induction of anoxia (Donnelly et al, 1992; Roberts Jr. et al, 1998). During anoxia, this dramatic membrane depolarization may activate a voltage-sensitive proton conductance (gu+), which produces 183 an outward proton current and a rise in pH i 5 as has been described in snail neurons and a variety of peripheral cell types (Meech and Thomas, 1987; Gordienko et al, 1996; Kapus et al, 1993). In addition, the fall in pHj induced by anoxia would act to lower the threshold voltage for activation of gH+ (Lukacs et al, 1993). Furthermore, the anoxia- evoked increase in [Ca 2 4 ! observed in the present study is likely consequent upon depolarization (Friedman and Haddad, 1993) and an elevation in [Ca ]j has been shown to potentate g^ in eosinophils, suggesting that the anoxia-evoked increase in [Ca2 +]j may also contribute to a significant activation of gH+ (Gordienko et al, 1996). Finally, Na7H + exchange can couple to H + conductances, such that inhibition of Na7H + exchange, as occurs during anoxia, can potentiate the outward proton conductance (Demaurex et al, 1995). Thus, activation of gH+ is entirely likely to occur during an anoxic period. Although the possible presence of a gH+ in rat hippocampal neurons has not been investigated, in all cell types studied to date sub-millimolar concentrations of ZnCl 2 effectively inhibit gH+ (DeCoursey and Cherny, 1994). In the present study, the addition of 500 p M ZnCl 2 reduced the magnitude of the pH, rise seen during a 10 min period of anoxia, suggesting the possibility that an outward H + flux may underlie the rise in pH; observed during anoxia. Furthermore, g H + is regulated by pH 0 ; an increase in pH 0 stimulates, while a decrease in pH 0 inhibits, current flow through these channels (Lukacs et al, 1993). This sensitivity to pH 0 may reflect a change in the concentration gradient for protons and charge screening, although the binding of protons to an allosteric extracellular site is also postulated to modify the activation properties of this conductive pathway (Lukacs et al, 1993; DeCoursey and Cherny, 1994). In the present study, there s 184 was a greater rise in pHj during anoxia under extracellular alkaline conditions and a smaller rise in pH; under extracellular acidic conditions, compared to the results obtained at pH 0 7.35. These results once again support the potential involvement of a in mediating the rise in pH, observed during anoxia, although the role of changes in pH ; consequent upon changing pH 0 in the regulation of gH+ is unknown. The decreased magnitude of the rise in pH, observed during anoxia at 22°C compared to 37°C may reflect the high temperature sensitivity of gH+, which produces a larger current at more elevated temperatures (Byerly and Suen, 1989), although a corresponding reduction in cerebral metabolic rate and, thus, an increase in the latency to anoxic depolarization cannot be excluded (Bart et al, 1998). Physiologically, the activation of gu+ may allow for the rapid alleviation of intracellular acidosis evoked by anoxia (DeCoursey and Chemy, 1994) and, thus, may play a role in pH ; regulation during an anoxic insult. Arguing against an involvement of g^ in the rise in pHj during anoxia are the facts that neither the removal of extracellular N a + nor the addition of 1 p M T T X influenced the relative magnitude of the per-anoxic recovery. Although these maneuvers are able to delay the onset of and/or reduce the magnitude of the rapid depolarization evoked by anoxia in rat hippocampal slices (Tanaka et al, 1997; Yamamoto et al, 1997), in the present study, neither influenced the time to onset of the rise in pH s observed during the anoxic period (data not shown). However, during an hypoxic insult in carotid body chemoreceptor cells, the ability of T T X to inhibit voltage-dependent N a + channels is inversely proportional to the severity of the membrane depolarization induced by hypoxia (Rocher et al, 1994). A similar situation may occur under reduced-Na+ 185 conditions. Thus, the possibility remains that voltage-sensitive proton channels are activated during anoxia and may contribute to the rise in pHj observed in adult rat hippocampal neurons. 5.2 Role of lactate transport during anoxia Secondary to lactate accumulation during an anoxic period, the efflux of lactate contributes to the extracellular acidification observed during an ischemic/anoxic insult (Siesjo et al, 1985). Moreover, carrier-mediated lactate transport has been described in hippocampal neurons, and the extrusion of lactate is associated with an intracellular alkalinization (Assaf et al, 1990; Kauppinen and Williams, 1990; Nedergaard and Goldman, 1993; Pirttila and Kauppinen, 1994b). Thus, with lactate accumulating intracellularly during an anoxic period (see Section 4.2), extrusion of lactate via lactate/Ff cotransport may contribute to the rise of pHj observed during an anoxic period. In support of this possibility, in the present study, there was a larger rise in pHj during anoxia in the presence of 17.5 m M glucose than in the presence of 5.0 m M glucose, a finding consistent with the fact that lactate accumulation, and thus lactate efflux, varies with pre-ischemic glucose concentrations, (Ljunggren et al, 1974). Studies examining neuronal lactate/Ff cotransport have failed to observe any significant effect of 4a-5-hydroxycinnamate (a-CHC), an effective lactate/Ff cotransport inhibitor in peripheral cell types, on either the rate of lactate uptake or its extrusion in mammalian central neurons and glia (Walz and Mukerji, 1988; Nedergaard and Goldman, 1993; Pirttila and Kauppinen, 1994b; but see Dringen et al, 1993). Similarly, in 186 preliminary studies, 5 m M a-CHC fails to affect resting pH; in acutely dissociated adult rat hippocampal neurons (data not shown). While previous reports in neurons have discounted the contribution of lactate/H+ cotransport to pH ; regulation during anoxia (Kauppinen and Williams, 1990; Prittila and Kauppinen, 1992), the present study is unable to do so. Following an ischemic period, the removal of accumulated intracellular lactate is central to the recovery of pHj in cardiac tissue. (Vandenberg et al, 1993). In the present study, given that the magnitude of the post-anoxic alkalinization was not influenced by reducing extracellular glucose concentration from 17.5 to 5 m M glucose, lactate/H+ cotransport is unlikely to play a major role, i f any, in the recovery of pH ; following a transient period of anoxia in rat hippocampal neurons. Indeed, in cortical brain slices, lactate transport proceeded at a significantly slower rate than p i t recovery following anoxia (Pirttila and Kauppinen, 1992). In summary, the results of the present study suggest, albeit indirectly, the presence of two additional pHj regulating mechanisms in adult rat hippocampal neurons, namely, a voltage-dependent proton conductance and/or a lactate/H+ cotransporter. Furthermore, these mechanisms may contribute to the rise in pHi observed during anoxia and, in doing so, may help to counteract a potentially harmful profound decline in pHj. 6 Factors Contributing to the Post-Anoxic Alkalinization Following a transient anoxic period, there was an intracellular alkalinization above the initial pre-anoxic, steady-state pH; from which the majority of cells did not 187 show any recovery. Intracellular pF^ "overshoots" have been observed following ischemia (both in vivo and in slice preparations in vitro), acute glutamate application, and induced seizure activity (Mabe et al, 1983; Siesjo et al, 1985; Yoshida et al, 1985; Pirttila and Kauppinen, 1992; Hartley and Dubinsky, 1993; Ou-Yang et al, 1995), although in none of these cases have the underlying mechanism(s) been identified. 6.1 Role of a voltage-activated proton conductance One possible factor which may contribute to this phase of the pH, response to transient anoxia is a persistent neuronal depolarization which, in turn, activates proton efflux through voltage-dependent H + channels. Once anoxic depolarization occurs, the return to normoxia is not necessarily associated with membrane repolarization, and, in fact, a further depolarization may be seen (Tanaka et al, 1997; Yamamoto et al, 1997). Increasing the duration of anoxia not only increases the likelihood of anoxic depolarization, but also decreases the likelihood of repolarization upon the return to normoxia (O'Reilly et al, 1995). In the present study, [Ca2+]i remained elevated in the post-anoxic period, suggesting that repolarization does not occur (e.g. Silver and Erecinska, 1990; Tanaka et al, 1997; Pisani et al, 1998). Furthermore, 500 p M Zn 2 + reduced the magnitude of the post-anoxic alkalinization following a 10 min period of anoxia. Therefore, a gH+ active in the immediate post-anoxic period may contribute to the intracellular alkalosis observed following a transient anoxic insult in adult rat hippocampal C A I neurons. 188 6.2 Changes in NaVFT exchange activity In cardiac myocytes, the Na7H + exchanger is activated immediately upon reperfusion following an ischemic/anoxic insult (Maddaford and Pierce, 1997) and is primarily responsible for both the post-anoxic increase in [Na+]( and the recovery of pHj from the intracellular acidosis produced during the anoxic period (Tani and Neely, 1989; Docherty et al, 1997). In the present study, substituting extracellular N a + with N M D G + (a maneuver which inhibits the Na7H + exchanger) attenuated the magnitude of the post- anoxic alkalinization, whereas, when L i + was employed as the extracellular Na + substitute, this phase of the pHj response was restored. These findings suggest that the Na7H + exchanger in rat hippocampal neurons is activated immediately following the return to normoxia and contributes to the rise in p l i observed after the anoxic period. This possibility was confirmed directly by comparing the rates of pHj recovery from acid loads imposed under HC03"-free conditions both prior to and following anoxia. Following a transient anoxic period, there was an increase in Na7H + exchange activity, along with an alkaline shift in its pHj sensitivity. The present results are consistent with previous reports which have suggested that activation of Na7H + exchange activity may participate in the restoration of pit; following the intracellular acidosis evoked by anoxia. Thus, in hippocampal slices, Pirttila and Kauppinen (1992) suggested that while the Na7H + exchanger was not functional during an anoxic period, its activity was central to the production of the post-anoxic overshoot in pHj. Furthermore, Obrenovitch et al (1990) suggested that the activation of Na7H + exchange contributes to the extracellular acidosis observed following an anoxic period. 189 And, in cultured fetal cortical neurons, Vornov et al (1996) employed pharmacological inhibitors to similarly conclude that inhibition of NaVFf exchange decreases the rate of recovery of pH ; following a transient period of metabolic inhibition. However, none of these studies directly assessed Na7H+exchange activity in the post-anoxic period. 6.2.1 Possible factors contributing to the increase in Na+/E? exchange activity Given that there is a significant activation of Na7H + exchange activity in the post- anoxic period, the relevant issue then becomes the identity of the underlying activating factor(s). One factor that could potentially contribute to the increased Na + /H + exchange activity following an anoxic insult is a reduction in intrinsic buffering power (Pi). However, present estimates of p ; under normoxic and post-anoxic conditions, at pH 0 7.35 and over a similar pHj range, do not support this possibility. Moreover, over a range of pH 0 values and temperatures, there was no change in PJ prior to or following an anoxic period. Thus, the observed increase in the rate of pH, recovery following an anoxic period appears to be unlikely to reflect a decrease in PJ . Secondly, in cardiac myocytes in vivo, the intracellular acidification produced during anoxia and the rapid normalization of pH 0 which occurs in the immediate post- anoxic period act to stimulate Na7H + exchange activity (Tani and Neely, 1989; Maddaford and Pierce, 1997). However, corresponding changes in pH ; and pH 0 are not the only factors which promote the post-anoxic activation of the Na7H + exchanger in rat hippocampal neurons because: i) pH 0 was kept constant throughout the experiment and 190 yet Na7H + exchange activation still occurred upon the return to normoxia; and ii) in some cases, pH; recovered to pre-anoxic steady-state levels in the continued absence of oxygen (e.g. Fig. 8B), but, once again, post-anoxic activation of the Na7H + exchanger occurred. Thirdly, the sustained increase in lactate levels which occurs in the immediate post-anoxic period (Kobayashi et al, 1977; Hope et al, 1988; Kauppinen and Williams, 1990) may stimulate Na7H + exchange activity. The metabolism of lactate generates protons, whose intracellular accumulation activates Na7H + exchange, thereby contributing to the production of the post-anoxic alkalinization (Mabe et al, 1983; Katsura et al, 1992). Indeed, the detrimental effect of lactate accumulation on cellular recovery following an ischemic period in cardiac myocytes is reportedly mediated through a post-anoxic stimulation of Na7H + exchange (Karmazyn et al, 1993). However, in the present study, there was no difference in the magnitude of the post-anoxic alkalinization produced in media containing 17.5 or 5.0 m M glucose, conditions in which the intracellular levels of lactate will vary. Finally, changes in cellular energy state and the activities of intracellular second messenger systems which act to control the activity of the Na7H + exchanger may contribute to the activation of the exchanger in the immediate post-anoxic period. The possibility that a rapid restoration of intracellular ATP may trigger or permit Na7H + exchange activation has already been discussed (see Section 4.3). A n additional possibility is that anoxia-evoked changes in the activities of intracellular second messenger systems may produce the observed increase in Na7H + exchange activity. Thus, in hippocampal neurons, stimulation of the c A M P / P K A pathway causes both an 191 increase in the slope and a large alkaline shift in the x-intercept of the linear regression line relating rates of pHj recovery to absolute pH ; (Smith et al, 1998). These changes in Na7H + exchange activity are analogous to those observed in the present study following a transient period of anoxia, raising the possibility that the increase in the activity of the NaVFf exchange activity in the post-anoxic period may be consequent upon stimulation of the c A M P / P K A pathway. Indeed, during recovery from an ischemic insult, cAMP levels rise rapidly (Kobayashi et ql, 1977; Domanska-Janik and Pylova, 1989; Blomqvist et al, 1985) and stimulus-evoked accumulations of [cAMPjj are enhanced (Domanska- Janik and Pylova, 1989). Furthermore, in the present study, the addition of Rp-cAMPS, a P K A inhibitor, significantly decreased the magnitude of the post-anoxic alkalinization. On the other hand, the addition of P-adrenoceptor agonists, which stimulate the c A M P / P K A pathway, significantly increased the magnitude of the post-anoxic alkalinization. Thus, a post-anoxic increase in [cAMP] ; may contribute to the post-anoxic activation of NaTFf exchange in rat hippocampal neurons. As will be discussed in the following section (Section 6.3), just as an increase in NaVFf exchange activity following anoxia promotes cellular dysfunction, an increase in [cAMP], is also apparently correlated with an increased likelihood of neuronal damage following anoxia/ischemia (Domanska-Janik and Pylova, 1989; Shibata et al, 1992). Thus, the intracellular cascade of increased NaVFf exchange activity in the post-anoxic period promoting neuronal damage may be due to an increased activity of the c A M P / P K A second messenger system. 192 6.3 Potential physiological relevance of the post-anoxic activation of~NdV7T exchange In peripheral cell types, for example cardiac myocytes, it is well established that Na7H + exchange inhibition effectively protects against reperfusion-induced cellular injury following ischemia (reviewed by Scholz and Albus, 1993; Piper et al, 1996; Bugge et al, 1996). In addition, in cultured fetal neocortical neurons, it has recently been reported that neuronal survival following metabolic inhibition (2-deoxy-D-glucose and cyanide) was improved when the Na7H + exchanger was inhibited (Vornov et al, 1996). However, the precise nature of the relationship between Na7H + exchange activity in the post-anoxic period and cellular viability remains unknown. On the one hand, Na7H + exchange activity following anoxia may cause an increase in intracellular Na + , which, in combination with membrane depolarization, could reverse the activity of the Na7Ca 2 + exchanger. Through activation of various proteases, endonucleases, and phospholipases, the resulting Ca 2 + overload may ultimately initiate cell death (Choi, 1990; Herman et al, 1990; Arai et al, 1991). Thus, inhibition of Na7H + exchange in the immediate post-anoxic period may reduce potentially harmful increases in [Na+]i and [Ca ]j following anoxia (Tani and Neely, 1989). On the other hand, recent studies have suggested a role of Na7H + exchange, independent of changes in [Ca 2 +] i 5 in the regulation of cellular viability, indicating that the normalization of pH ; following a transient anoxic period per se promotes cell death (Bond et al, 1993), and that cytoprotection does not necessarily require the attenuation of a post-anoxic increase in [Ca2+]i (Harper et al, 1993). In support of a dissociation between Na7H + and reverse 193 Na7Ca 2 + exchange activity following anoxia, inhibition of NaTFf exchange in isolated cardiac myocytes blocks the apparently deleterious restoration of pH; in the post-anoxic period while not influencing the rise in [Ca2+]j (RuP et al, 1996). A role for NaTFf exchange in neuroprotection, independent of changes in [Ca2 +] i 5 has also been suggested in spinal cord axons, where NaTFf exchange inhibitors improved, and NaTCa 2 + exchange inhibitors did not improve, functional recovery following spinal cord compression (Agrawal and Fehlings, 1996). Similarly, in the present study, the anoxia-induced cellular injury that occurred during and following a transient anoxic period (as indicated by membrane bleb formation and swelling) continued to occur in the absence of 2+ 2+ extracellular Ca , a condition which attenuated the anoxia-evoked increases in [Ca ]; (also see Friedman and Haddad, 1994a), suggesting that an increase in [Ca \ is not an absolute prerequisite for acute damage in response to anoxia in acutely dissociated hippocampal neurons. Thus, the post-anoxic increase in NaTH + exchange activity observed in the present study may promote neurodegeneration following anoxia, not by modulating [Ca 2 +] i 5 but by increasing in [Na+]i and decreasing [H+][. A decrease in [H +] ; activates the majority of degredative enzymes (Herman et al, 1990). Additionally, overactivity of NaTH + exchange, and influx of N a + through voltage-sensitive Na + channels, will increase [Na+]j and may contribute to a worsening of cellular energy state 2+ (Erecinska and Dagani, 1990), a reduction in Ca extrusion, and an initiation of early osmotic injury (Jakubovicz and Klip, 1997). 194 6.3.1 The modulatory effects of changes in extracellular pH Profound shifts in extracellular pH occur during and following transient ischemic periods in vivo and in slice preparations in vitro (Mutch and Hansen, 1984; Siesjo et al, 1985; Erecinska and Silver, 1994). As noted in the Introduction (see Section 3.2), these changes in pH 0 may have a direct influence on neuronal survival. Indeed, following an ischemic insult, an increase in pH 0 increases the susceptibility of neurons within the penumbral area to damage (Choi, 1990). Conversely, mild extracellular acidosis exerts a neuroprotective effect attributed to reduced NMDA-mediated excitotoxicity, reduced 2+ + intracellular Ca and Na overload, and the suppression of neuronal excitability and a consequent reduction in metabolic energy demand (Giffard et al, 1990; Krnjevic and Walz, 1990; Tombaugh and Sapolsky, 1990; Cummins et al, 1991; Ebine et al, 1994; Ou- Yang et al, 1994a; Tombaugh and Somjen, 1996; Vomov et al, 1996). Given the relationship between the post-anoxic activation of Na7H + exchange and neuronal survival (see Section 6.3), the results of the present study indicate that the established neuroprotective effects of extracellular acidosis and the detrimental effects of extracellular alkalosis may also reflect their influence on the absolute level of Na7H + exchange activity in the immediate post-anoxic period. Thus, the rate of Na7H + exchange activity in the post-anoxic period was significantly faster at pH 0 7.60 and significantly reduced at pH 0 6.60. As pH 0 was increased, there was a progressive increase in the slope, and alkaline shift in the x-intercept, of the linear regression lines relating pHj and rates of pHj recovery from intracellular acid loads imposed following an anoxic period. Thus, the 195 ability of changes in pH 0 to influence neuronal viability following a transient anoxic period may be mediated, at least in part, by changes in the degree of post-anoxic activation of Na7H + exchange. 6.3.2 The modulatory effects of changes in ambient temperature Mild hypothermia prevents neuronal loss of function that occurs in response to anoxia (Kurihara et al, 1996; Taylor and Weber, 1993). In fact, hypothermia may be a behavioral and/or physiological response to oxygen deprivation (Wood and Gonzales, 1996). The neuroprotection afforded by mild hypothermia has been suggested to be related to a reduction in cerebral energy utilization rate and a corresponding attenuation of anoxia-induced reductions in ATP levels (Kozlowski et al, 1997; Bart et al, 1998). The results of the present study suggest an additional mechanism whereby hypothermia may exert a neuroprotective effect, i.e. a reduced activity of the Na7H + exchanger in the immediate post-anoxic period. Thus, the present data not only support the role of the Na7H + exchanger in the production of the post-anoxic alkalinization, but also uphold the relationship between Na7H + exchange and neuronal viability. 7 Summary This study detailed the pH; response of acutely isolated adult rat hippocampal CA1 neurons to transient anoxic periods of varying durations, and assessed the mechanisms which contribute to the production of each of the pHj shifts which occurred. As supported by evidence presented in this study, Figure 39 outlines the pHj regulating 196 mechanisms that may underlie the pH; response to a transient period of anoxia in this cell types. While supporting the role of ATP hydrolysis and lactate accumulation in the production of the fall in pHj during anoxia, the present study suggests that inhibition of the acid-extruding NaTFT exchanger during anoxia may also contribute to the production of the intracellular acidification produced during the anoxic period. The gradual rise in pHj seen in the continued absence of oxygen was tentatively ascribed to the activation of Zn2+-sensitive, voltage-dependent proton conductance and/or lactate/Ff cotransport. Finally, a rapid and significant activation of the NaTFf exchanger was found to be responsible, in large part, for the post-anoxic alkalinization, although membrane depolarization may also contribute to this phase of the pH ( response. Given the ability of pHj to influence many facets of neuronal function, the anoxia-evoked shifts in pHj and changes in the activities of pHj regulating mechanisms may determine, at least in part, neuronal viability following a transient period of oxygen deprivation. 197 Figure 38. Effect of a 5 min period of anoxia on [Na+]( The experiment was performed under HC0 37C0 2-free, HEPES-buffered conditions. Prior to the induction of anoxia, [Na+]j was ~ 25 mM. Upon the induction of anoxia, [Na+]j began to increase after ~ 0.80 min, reaching a maximum value of ~ 70 m M at the end of the anoxic period. Upon the return to normoxia, [Na+]j increased further to 100 mM. Changes in [Na+]; were measured using SBFI (sodium-binding benzofuran phthalate), a sodium-sensitive ratiometric fluorophore. Acutely dissociated hippocampal CA1 neurons were loaded with 30 p M of the AM-ester form of SBFI for one hour at 37°C. SBFI was excited at wavelengths 334 nm and 380 nm and the emitted wavelengths were measured at 520 nm. In an analogous situation to that performed for BCECF, a one- point calibration was performed at the completion of the experiment. This one-point calibration solution contained 10 m M N a + and 5 p M gramicidin, a N a + ionophore. A full calibration curve was obtained during experiments in which cells were exposed to [Na+]0 ranging between 0 m M to 130 mM. Time (min) 199 Figure 39. pH { regulating mechanisms that may contribute to the anoxia-evoked changes in pH, observed in acutely dissociated adult rat hippocampal CAI neurons in response to transient periods of anoxia This diagram outlines the mechanisms discussed in the present study that may contribute to the characterized pPf response to transient periods of anoxia. The study supports the contribution of ATP hydrolysis and lactate accumulation in the production of the intracellular acidification observed during an anoxic period. In addition, this study provides direct evidence for inhibition of NaTFf exchange activity during anoxia which may also contribute to this acidification. Anoxic depolarization may activate a voltage- sensitive proton conductance which, in turn, may contribute to the rise in pH; observed in the continued absence of oxygen. Lactate/Ff cotransport may also contribute to this phase of the pHj response. 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