Cyclic AMP Concentrations in Rat Neocortex and Hippocampus During and Following Incomplete Ischemia: Effects of Central Noradrenergic Neurons, Prostaglandins, and Adenosine

The concentrations of cyclic AMP, noradrenaline, glycogen, glucose, lactate, pyruvate, labile phosphate compounds, and free fatty acids were investigated in the rat neocortex and hippocampus during and following cerebral ischemia. An incomplete ischemia of 5 and 15 min duration was induced by bilateral carotid clamping combined with hypotension. The postischemic events were studied after 5, 15, and 60 min of recirculation. Five minutes of ischemia did not significantly alter the neocortical or hippocampal concentrations of cyclic AMP. After 15 min of ischemia the neocortical levels decreased significantly below control values. In the recirculation period following ischemia a significant elevation of the cyclic AMP concentrations was observed. Following 5 min of recirculation after 5 min of ischemia the levels increased from 2.53 +/- 0.21 nmol X g-1 to 5.18 +/- 0.09 nmol X g-1 in the neocortex and from 2.14 +/- 0.16 nmol X g-1 to 3.52 +/- 0.35 nmol X g-1 in the hippocampus. Five minutes of recirculation following 15 min of ischemia led to a significant increase in the levels of cyclic AMP, to 12.86 +/- 1.43 nmol X g-1 in the neocortex to 5.58 +/- 0.57 nmol X g-1 in the hippocampus. With longer recirculation periods the cyclic AMP levels progressively decreased and were similar to control values after 60 min. Depletion of cortical noradrenaline by at least 95% was performed by injections of 6-hydroxydopamine into the ascending axon bundles from the locus ceruleus. The lesion did not significantly change the ischemic or post-ischemic neocortical and hippocampal levels of cyclic AMP, glycogen, or free fatty acids including arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of low intracellular or extracellular pH in sensitization to hyperthermia

Cells are more sensitive to heat when they are heated in an acidic environment, and this study confirms (K. G. Hofer and N. F. Mivechi, J. Natl. Cancer Inst., 65, 621, 1980) that intracellular pH (pHi) and not extracellular pH (pHe) is responsible for the sensitization. The relationship between pHe, pHi, and heat survival of cells heated in vitro in various buffers at pHe 6.3-8.0 was investigated. Cells' adaptation to low environmental pH in terms of increases in pHi and heat survival also was investigated. Finally, we studied the relationships among pHe, pHi, and survival from heat for cells heated in sodium-free reconstructed medium. Intracellular pH was measured by the distribution of the weak acid, [2-14C]5,5-dimethyl-2,4-oxazolidinedione. Our results are summarized as follows: (1) CHO cells maintained the same relationship between pHe and pHi in four different media or buffers (McCoy's 5a medium buffered with CO2 and NaHCO3 or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic acid (Mes), Krebs-Ringer bicarbonate solution, and Krebs-Ringer phosphate solution) with pHi being 0.05 to 0.20 pH units higher than pHe as it varied from 7.0 to 6.4; furthermore, heat sensitization by acid was the same in medium buffered with NaHCO3 or Hepes and Mes. (2) The low pHe adapted cells multiplied with an increased doubling time of 20.7 +/- 0.7 h and appeared morphologically similar to the unadapted cells. However, the pHi of these cells was 0.15-0.30 pH units higher than that of the unadapted cells when pHe was varied between 7.0 and 6.3. (3) After being heated at 43.5 degrees C for 55 min or at 42.5 degrees C for 150 min at pHe 6.3-7.2, the pHi of the adapted cells increased by 0.2-0.1 pH units. However, heat caused no significant change in the unadapted cells. (4) Heat survival plotted versus pHe was 1000-fold higher for the adapted cells than for the unadapted cells at pHe of 6.3. However, heat survival plotted versus pHi was identical for the two cell types. (5) In sodium-free reconstructed McCoy's 5a medium, pHi was 0.25-0.1 pH units lower than that in the sodium-containing counterpart at pHe 6.3-7.2, and heat sensitization increased accordingly; however, heat survival plotted versus pHi was identical for the two types of media.     

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.     

Effect of hyperthermia on intracellular pH in Chinese hamster ovary cells

Chinese hamster ovary cells were heated at 45.5 or 43.0 degrees C at acidic pH (6.7) or normal physiological pH (7.4) to have a survival of 10(-3). The weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C), was used to measure the intracellular pH (pHi) both during and following hyperthermia. Tritiated water and a Particle Data machine were used to measure cellular volume as well. With 99.9% of the cell population destined to die clonogenically, the physiologically alive cells, as determined by the exclusion of trypan blue dye, maintained their pH differential between pHe and pHi as well as unheated cells. Furthermore, the cell's ability to regulate its pHi in response to changes in pHe was not affected by the same hyperthermic treatment. However, cellular volume decreased by 15-30% by 5 h after the onset of heat treatment. We conclude that heat does not perturb the cellular regulation of intracellular H+ concentration. Therefore, there is no thermal damage to the pHi-regulatory mechanism that could be responsible for either heat-induced reproductive cell death or low pH sensitization of heat killing.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Intracellular pH, Lactate, and Energy Metabolism in Neonatal Brain During Partial Ischemia Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Sequential 31P and 1H nuclear magnetic resonance spectra were measured for neonatal piglets (n = 7) to determine the relationship between brain intracellular pH (pHi), lactate, and phosphorylated energy metabolites during partial ischemia. Simultaneous determinations of arterial and cerebral venous blood gases, pH, O2 content, and plasma concentrations of glucose and lactate were also made. Ischemia, induced by bilateral carotid artery ligation plus hemorrhagic hypotension for 35 min, resulted in variable reductions in ATP, phosphocreatine, and increases in Pi, H+, and lactate relative to control levels. In four piglets, whose arterial blood glucose rose above control, brain lactate exceeded 20 mumol g-1 with corresponding decreases in pHi of greater than 0.7 units compared to control levels. The extents of brain acidosis and lactosis showed a strong linear correlation with each other (r = 0.94). Maximal changes in brain lactate, pHi, and ATP at the end of ischemia showed significant positive linear correlations with the control levels of arterial blood glucose, but did not correlate with arterial glucose or arterial cerebral-venous glucose difference values during ischemia. The relationship between pHi and buffer base deficit was comparable to results reported for adult animals up to 20 mumol ml-1. However, in contrast to models proposed for adult brain, the continued linear relationship between pH and higher buffer base levels is most consistent with a theoretical model that assumes the presence of weak acid buffers with pKa values from 6.7 to 5.2.     

Cell volume regulatory responses of isolated perfused rat liver. The effect of amino acids

1) Addition of glutamine, glycine, alanine, serine, phenylalanine, proline at a concentration of 3mM, each, or of an amino-acid mixture resembling the physiological amino-acid composition of portal venous blood, to influent perfusate of isolated perfused rat liver led to a 4-6% increase of liver mass without increase of the [3H]inulin space, and biphasic K+ movements across the plasma membrane. These K+ movements consisted of an initial net K+ uptake (0.4-0.9 mumol X g-1 liver) for about 2 min, being followed by a net K+ release (1.0-2.8 mumol X g-1 liver) during the next 10 min. Withdrawal of the amino acids from influent perfusate caused a slow net K+ reuptake by the liver and restored the initial liver mass. No effects on liver mass and K+ fluxes were observed following addition of glutamate or glucose at a concentration of 3mM, each. 2) Aminooxyacetate did not affect the alanine (3 mM) induced increase in liver mass. However, in presence of aminooxyacetate the alanine-induced net K+ release from the liver (i.e. K+ release from 2-10 min minus initial K+ uptake) increased from 0.1 to 2.2 mumol X g-1 liver, whereby simultaneously the alanine tissue level rose from 6.8 to 13.3 mumol X g-1 (corresponding to an increase of the intracellular alanine concentration from about 12 to 25 mM) in presence of aminooxyacetate. 3) When livers were perfused with different glutamine concentrations, a maximal increase in liver mass of 5-6% was observed at glutamine concentrations above 1.5-2mM. A halfmaximal increase in liver mass was observed at 0.6-1.0mM glutamine in influent, i.e. at the physiological portal glutamine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Extracellular and intracellular acid-base status with regard to the energy metabolism in the oyster Crassostrea gigas during exposure to air

The acid-base status of extra- and intracellular fluids was studied in relation to the anaerobic energy metabolism in the adductor muscle, mantle, gills, and heart of the marine bivalve Crassostrea gigas after exposure to air for periods of 2, 4, 8, 12, 24, and 48 h. Such exposure was found to cause a significant reduction in the pH in the hemolymph (pH(e)) within the first 4 h. The decrease in the pHe was accompanied by elevated Pco2 values, causing [HCO3-] to rise (respiratory acidosis). Thereafter, the pHe fell at a lower rate, and this fall was partially compensated for by a further increase in [HCO3-] in the hemolymph. The increase in the [Ca] levels in the hemolymph indicates a mobilization of Ca2+ from CaCO3 and the involvement of bicarbonates in the buffering of pHe. The main anaerobic end-products that accumulated in the tissues during the first stages of anaerobiosis were alanine and succinate, at a ratio of about 2 : 1. Later on, propionate and acetate were also accumulated at significant rates. In contrast to the adductor muscle, gills, and mantle, opine production in the heart was significant after 12-24 h of exposure to air. Determination of intracellular pH (pHi) revealed that there is a close relationship between the rate of anaerobic end-product accumulation and the extent of intracellular acidosis in the adductor muscle, mantle, and gills. On the contrary, accumulation of anaerobic end-products in the heart did not cause any significant change in its pHi. The intracellular nonbicarbonate, nonphosphate buffering value (beta (NB,NPi)) was determined to be higher in the heart than in the other three tissues and thus probably plays a crucial role in stabilizing heart pHi during exposure to air.     

The presence of HCO3- does not inhibit alpha-adrenergic increases in proximal tubular intracellular pH

Hormonal stimulation of Na+/H+ exchange increased intracellular pH (pHi) in a dose-dependent manner in proximal tubules suspended in Krebs-Henseleit buffer (KHB) supplemented with 25 mM HCO3- and CO2 (KHB + HCO3). The maximum increase in pHi was approximately 45% of the response observed with segments suspended in bicarbonate-free buffer (KHB-HCO3) and the time required to achieve maximum pHi alterations was significantly increased (p less than 0.05) in the presence of KHB + HCO3 when compared to responses obtained in KHB - HCO3. Dose-response curves for agonist-induced pHi increases were shifted to the right by a factor of 10 for segments suspended in KHB + HCO3. Increases in pHi induced by agonists in KHB + HCO3 were effectively blocked by pretreatment with 10 microM ethylisopropyl amiloride but not with the Cl-/HCO3- inhibitor, DIDS (0.1 mM, 30 min). We conclude that stimulation of alpha-adrenergic receptors on proximal nephrons increased pHi due to activation Na+/H+ exchange and can be detected in the presence of HCO3- although the time course and maximum level of change differ significantly from those observed in a HCO3(-)-free buffer.     

Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions.

The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5,5'-dimethyloxazoldine,-2,4-dione (DMO) method. The initial pHi at 4degrees was about 7.6 and was higher than the extracellular pH (pHe) at 4degrees. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4degrees) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2,3-diphosphoglycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4degrees were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.     

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.     

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of basolateral CO2/HCO3- on intracellular pH regulation in the rabbit S3 proximal tubule

We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D- glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Partial Restoration of Cerebral Myelination of the Congenitally Hypothyroid Mouse by Parenteral or Breast Milk Administration of Thyroxine

The objective of the present study was to assess metabolic changes in the neocortex and hippocampus of well-oxygenated or moderately hypoxic rats in which fluorothyl-induced seizures were sustained for 5 or 20 min, or which were allowed recovery periods of 5, 15, or 45 min following cessation of 20-min seizure activity by withdrawal of the convulsant gas. Sustained fluorothyl-induced seizures were found to cause metabolic alterations qualitatively and quantitatively similar to those previously observed with other commonly used convulsants. Thus, although the phosphorylation state of the adenine nucleotide pool remained only moderately perturbed, if at all, there were decreases in tissue concentrations of phosphocreatine and glycogen, and increases in those of cyclic AMP, lactate, and pyruvate, with a calculated fall in intracellular pH of about 0.15 units and a rise in the cytoplasmic NADH/NAD+ ratio. The enhanced metabolic rate was reflected in a marked reduction in the tissue-to-plasma glucose concentration ratio. Induced moderate hypoxia (arterial PO2 40-50 mm Hg) had no metabolic effect after 5 min of seizures but moderately increased lactate concentrations after 20 min (from about 10 to about 15 mumol X g-1). On cessation of seizure discharge cyclic AMP and phosphocreatine concentrations normalized already within 5 min, whereas glycogen and lactate concentrations normalized more slowly. In the neocortex (but not the hippocampus) postepileptic tissue-to-plasma glucose concentration ratios rose above control, probably reflecting metabolic depression. The results suggest that intracellular pH promptly returned to control, and that postepileptic alkalosis developed. They also suggest that some elevation of the NADH/NAD+ ratio persisted even after 45 min of recovery.     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.