Dose-dependent metabolic response of mammary carcinoma to photodynamic therapy

The metabolic response of mammary carcinoma in the C3H mouse to photodynamic therapy (PDT) was measured using in vivo 31P nuclear magnetic resonance (31P-NMR) spectroscopy and pH microelectrodes. Twenty-four hours after administration of Photofrin II (12.5 mg/kg), the tumor was subjected to photoactivation using an argon dye laser. Optical treatment doses were 200, 400, and 600 J/cm2 and corresponded to the following tumor control doses: TCD10/30, TCD50/30, and TCD90/30, respectively. In vivo 31P-NMR spectra and pH micro-electrode measurements were obtained prior to treatment and at 4, 24, 48, and 72 h and 1 week post-treatment. The data revealed a significant (P less than 0.0002) alkalosis as indicated by the pH measured by NMR compared to pH measured by microelectrodes at all treatment levels and time points. Spectral differences between treatment groups were apparent as early as 4 h after treatment. The ratio of beta-nucleoside triphosphate to inorganic phosphate at 4 h after treatment was significantly (P less than 0.01) smaller for 600 J/cm2 treatment than for 200 J/cm2 treatment. At curative (600 J/cm2) levels, from 48 h on, no phosphate resonances were detected in the spectra. The pH measured by NMR transiently decreased from pretreatment levels after 200 and 400 J/cm2 treatment (P less than 0.002, P less than 0.009, respectively), while no change in pH from pretreatment values was found after 600 J/cm2 treatment. The data suggest that the early metabolic response of mammary carcinoma to PDT, as indicated by 31P-NMR spectroscopy, is dose dependent, and may be a sensitive indicator of biological outcome to treatment.     

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.     

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.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Changes in muscle pH following hyperthermia

Considerable attention has been paid to the modification of intratumor pH in response to hyperthermia. It has been hypothesized that observed reductions in intralesional pH are involved in the ultimate response of tissue to hyperthermia treatment. Further, it has been shown that significant differences exist in hyperthermia-induced changes in blood flow between tumor and normal tissue in many systems. Changes in blood flow are hypothesized to be related to observed changes in pH. Since reduced blood flow is not observed in normal tissue under normal treatment conditions, changes in pH in normal tissue have not been considered significant in their response to hyperthermia treatment. However, this conclusion has not been verified or documented experimentally. The purpose of this study was to examine the distribution of pH in normal tissue (muscle) as a function of time following hyperthermia treatments which in the same animal system resulted in subcurative (TCD 10/30) or curative (TCD 90/30) tumor (mammary adenocarcinoma) responses. The observed distribution of pH in normal tissue was compared with that obtained in tumors under identical conditions. The results indicate that some post-treatment changes in muscle pH do occur following hyperthermia, but that these changes are small compared to those observed in tumors. More importantly, unlike the response observed in tumors, no hyperthermia dose dependency is observed in the muscle response. From these studies it can be concluded that changes in normal muscle pH are probably not associated with normal tissue response to hyperthermia.     

Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity

The effect of variable extracellular pH on intracellular pH, cell energy status, and thermal sensitivity was evaluated in CHO cells over the extracellular pH range of 6.0 to 8.6. Extracellular pH was adjusted with either lactic acid, HCl, or NaOH. Regardless of the method of pH adjustment, the results obtained were similar. The relationship between extracellular and intracellular pH was dependent upon the pH range examined. Intracellular pH was relatively resistant to a change in extracellular pH over the pHe range of 6.8 to 7.8 (i.e., delta pHi congruent to delta pHe X 0.33). Above and below this range, delta pHi congruent to delta pHe X 1.08 or X 0.76, respectively. Cellular survival after a 30-min heat treatment at 44 degrees C remained constant over the extracellular pH range of 7.0 to 8.4, but varied substantially over a similar intracellular pH range. The cellular concentration of the high energy phosphate reservoir, phosphocreatine, decreased with decreasing pH. However, the cellular concentrations of ATP, ADP, and AMP remained constant over the entire pH range examined. It is concluded that increased thermal sensitivity resulting from a change in extracellular pH is not due to cellular energy depletion. Furthermore, intracellular pH is a more accurate indicator of thermal sensitivity than is extracellular pH.     

Variations in pO2 and pH response to hyperthermia: dependence on transplant site and duration of treatment.

It has been clearly established that changes in intratumor pO2 and pH occur following hyperthermia, and it has been hypothesized that these changes may, in some way, be related to the ultimate response (i.e., cure) of the lesion. The purpose of this study was twofold: first, to examine the changes in intratumor pH during the course of a hyperthermia treatment at biologically related end point "doses"; second, to examine the response of pO2 after treatment in a different lesion transplant site. During hyperthermia treatment of the tumor transplanted in the leg, intratumor pH was found to drop from a control value of 6.74 +/- 0.17 to 6.47 +/- 0.13 within 15 min following the start of treatment. The values then remained relatively constant throughout the remainder of the treatment (either 1 or 2 h at 43.5 degrees C). Following the subcurative (10% tumor cures at 30 days; 60 min at 43.5 degrees C) treatment the pH began to rise immediately, while after the higher dose (60% tumor cures at 30 days; 120 min at 43.5 degrees C) a slight rise in pH was followed by a continuous drop in pH for up to 4 h, as we have reported previously. Oxygen response in the two transplant sites (leg and flank) was found to be remarkably different even though the tumor cure rate was identical for a given hyperthermia "dose" in terms of time and temperature. In the leg, only very low levels of oxygen can be measured in the tumor 24 h after treatment with either "dose" studied (all measured pO2 values less than or equal to 5 mm Hg). In the flank, the tumor response is dependent on hyperthermia "dose." Only 28% of measured oxygen values are less than or equal to 5 mm Hg 24 h following a subcurative "dose," while 4 h following the higher "dose" there is a nonsignificant trend toward hypoxia (approximately 65% of values less than or equal to 5 mm Hg) with a subsequent shift toward reoxygenation. These latter observations are contrary to results reported previously and tend to contradict some current theories regarding the physiological mechanisms associated with hyperthermia treatment.     

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).     

pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.     

Dihydrotetrabenazine Binding and Monoamine Uptake in Mouse Brain Regions

The objective of the present study was to estimate extracellular pH (pHe) and intracellular pH (pHi) during near-complete forebrain ischemia in the rat, and to evaluate the relative importance of lactic acidosis and rise in tissue Pco2 (Ptco2) in causing pHe and pHi to fall. The animals, which were ventilated, normoxic, normocapnic, and normothermic, were subjected to 15 min of ischemia, either without or with 30-60 min of recirculation. Ptco2 was measured with a tissue electrode, pHe with a double-barrel liquid ion-exchanger microelectrode, changes in extracellular fluid (ECF) volume by impedance measurements, tissue CO2 content by a microdiffusion technique, and labile tissue metabolites by enzymatic fluorometric methods. Ischemia caused Ptco2 to rise to between 95 and 190 mm Hg (mean 149 mm Hg), and pHe to fall by 0.45-1.05 units (mean 0.70 units). During recovery, Ptco2 normalized within 5 min and pHe after 15-30 min. During ischemia, high-energy phosphates were depleted and tissue lactate content increased to 15 mumol X g-1. The total CO2 content (Tco2) was minimally or moderately reduced (normal, 11.9 mumol X g-1; range of ischemic values, 7.9-12.1 mumol X g-1), this range probably reflecting variable amounts of remaining blood flow. Impedance measurements demonstrated that ECF volume during ischemia was reduced to 55% of control, with gradual normalization during the first 15-30 min of recirculation. From values for Ptco2, Tco2, [HCO3-]e, and ECF volume, [HCO3-]i and pHi could be calculated. These values pertain to an idealized homogeneous intracellular compartment, and the methods used cannot detect whether different intracellular compartments diverge in their acid-base responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature and pH dependence of energy balance by (31)P- and (1)H-MRS in anaerobic frog muscle

The temperature (T)-dependence of energy consumption of resting anaerobic frog gastrocnemii exposed to different, changing electrochemical gradients was assessed. To this aim, the rate of ATP resynthesis (delta approximately P/deltat) was determined by (31)P- and (1)H-MRS as the sum of the rates of PCr hydrolysis (delta[PCr]/deltat) and of anaerobic glycolysis (delta[La]/ deltat, based on a approximately P/La ratio of 1.5). The investigated T levels were 15, 20 and 25 degrees C, whereas initial extracellular pH (pHe) values were 7.9, 7.3 and 7.0, i.e. higher, equal or lower, respectively, than intracellular pH (pHi). The latter was changing with T according to the neutrality point (dpH/dT=-0.0165 pH units/ degrees C). Both rates of PCr hydrolysis and of lactate accumulation and that of their sum, expressed as delta approximately P/deltat, were highly T-dependent. By contrast, the pHe-dependence of the muscle energy balance was nil or extremely limited at 15 and 20 degrees C, respectively, but remarkable at 25 degrees C (with a depression of the ATP resynthesis rate up to 25% with a decrease of pHe from 7.9 to 7.0). The pHe-dependent reduction of metabolic rate was associated with a down-regulation of anaerobic glycolysis due to reduced activity of ion-transporters controlling acid-base balance and/or to a shift from Na(+)/H(+) to a more efficient Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Uncoupling of glycogenolysis from P-metabolite concentrations, both as function of T (>or=20 degrees C) and of pHe (相似文献   

Stimulation-evoked changes in extracellular pH, calcium and potassium activity in the frog spinal cord

Double-barrel ion-sensitive microelectrodes were used to measure activity-related changes in extracellular pH (pHe), potassium and calcium concentration ([K+]e and [Ca2+]e) in the spinal dorsal horns of frogs. Repetitive stimulation (30-100 Hz) of the dorsal root evoked transient acidification in the lower dorsal horn by 0.25 pH units, which was accompanied by an increase in [K+]e by 4-5 mmol/l and a decrease in [Ca2+]e by 0.5 mmol/l. The pHe changes were found to have a typical depth profile and increased with the stimulation frequency, intensity and duration. The maximum of pHe changes was reached in 25-30 s of stimulation, and when stimulation continued further no greater pHe changes were achieved. Similarly as the K+ and Ca2+ transients, the pHe reached a ceiling level, which was 0.2-0.25 pH units more acid than the pH of the Ringer solution. The poststimulation K+ undershoot below the resting K+ level (3 mmol/l) was accompanied by an alkaline shift before the original pH base line. The rise time of the pHe changes was slower than that of [K+]e and [Ca2+]e changes. However, the redistribution of all the ionic changes had a similar time course. The clearance of changes in [K+]e and pHe was slowed by ouabain. The depression of the acid shift required higher concentrations of ouabain than the depression of the alkaline shifts. Acetazolamide, a carbonic anhydrase inhibitor, depressed the acid and enhanced the alkaline shift. Superfusion of the cord with elevated [K+]e was accompanied by a prompt and progressive acid shift, the lowering of [K+]e by an alkaline shift. The stimulus-evoked K+ increase and acid shift were depressed during the elevated [K+]e, while the alkaline shift was enhanced. Spontaneous elevations of [K+]e were accompanied by acid shifts of a similar time course. The results are discussed in terms of stimulus-evoked changes in extracellular strong ion differences [SID]e, and of their possible physiological significance.     

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.     

Effects of hypothermia on rat brain pHi and phosphate metabolite regulation by 31P-NMR

The effects of arterial alphastat regulation on brain intracellular pH (pHi) and several phosphate metabolites were assessed in anesthetized rats during hypothermia (28.6 +/- 0.2 degrees C) and normothermia (36.2 +/- 0.2 degrees C) by using 31P high-field (8.5 T) nuclear magnetic resonance (NMR). There were significant differences in pHi and metabolite ratios at the two temperatures under conditions of equal minute ventilation. During hypothermia, the brain pHi was 0.09 U higher, the phosphocreatine-to-inorganic phosphate (PCR/Pi) ratio 49% larger, and Pi-to-ATP 20% lower than at normothermia. These changes were fully reversible on warming the animal. The change in brain pHi/temperature was -0.011U/degrees C (95% confidence interval -0.007 to -0.016). The brain's ability to regulate its pHi and phosphate metabolism during hypercapnic acid-base stress was studied by using 10% CO2 ventilation. Hypothermic rats showed a larger fall in brain pHi (0.145 +/- 0.01 U, 7.15-7.01) with 10% CO2 than normothermic rats (0.10 +/- 0.02 U, 7.06-6.96). Similarly ventilated rats had a larger fall in arterial pH with 10% CO2 at hypothermia (0.36 +/- 0.04 U) than normothermia (0.24 +/- 0.01 U), so the delta brain pH/delta arterial pH was the same at both temperatures. The brain PCr-to-Pi ratio decreased approximately 20% during 10% CO2 breathing in both hypothermic and normothermic animals. Brain pHi and metabolite ratios returned to base line 30-50 min after CO2 washout in both groups. In summary, lowering body temperature while maintaining constant ventilation leads to changes in brain pHi and metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of inorganic phosphate and malate between intra- and extramitochondrial spaces. Relationship with the transmembrane pH difference

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.     

Effects of aluminum on the release and-or immobilization of soluble phosphate in corn root tissue

The effects of aluminum ions on the generation of mobile inorganic phosphate (Pi) within the cells of excised maize (Zea mays L.) root tips were examined using ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy. When perfused with a solution containing 50 mM glucose and 0.1–5.0 mM Ca²⁺ at pH 4.0, 3–5-mm-long excised maize root tips from 3-d-old seedlings showed a significant (approx. 100%) increase in the amount of mobile Pi, (primarily vacuolar) over a period of 30 h. This increase was above that which can be accounted for by the hydrolysis of endogenous sugar phosphates and nucleotides. A change of the pH of the perfusion solution to 7.0 reduced the increase in Pi to approx. 50%. Omission of Ca²⁺ in the solution at pH 4.0 caused the mobile Pi to increase to about 170%. However, the presence of Al³⁺ or both Ca²⁺ and Al³⁺ in the solution resulted in a significant loss (35–50%) of mostly vacuolar Pi over the same period of time. When root tips containing up to 65% of newly released Pi, produced after 20 h perfusion, were exposed to Al³⁺, no additional increase in the level of the mobile-Pi signal area was noted. Exposure to Al³⁺ with Ca²⁺ and glucose under hypoxia at pH 4.0 resulted in a threefold decrease in intracellular Pi content after the root tips were returned to aerobic conditions. These results indicate that external pH plays an important role in the generation of mobile intracellular Pi and that the presence of both Ca²⁺ and Al³⁺ can independently suppress the production of this excess Pi and ultimately reduce the vacuolar Pi.Abbreviations and symbols NMR nuclear magnetic resonance - Pi morganic phosphate - UDPG uridine diphosphoglucose - chemical shift     

A 31P NMR study of mitochondrial inorganic phosphate visibility: effects of Ca2+, Mn2+, and the pH gradient.

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (Pi) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix Pi is totally visible by NMR at 8 degrees C and at low external concentrations of Pi. However, when the external Pi concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix Pi increases, and the fraction not observed by NMR increases. Raising the temperature to 25 degrees C also decreases the pH gradient and the Pi fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix Pi NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of Pi signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the Pi peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix Pi should be mostly observable up to 25 degrees C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both Pi pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.     

Dual-function radiation sensitizers and bioreductive drugs: factors affecting cellular uptake and sensitizing efficiency in analogues of RSU 1069

Alkyl aziridine analogues of the hypoxic cell radiosensitizer RSU 1069 have been synthesized and one of these, RB 7040, containing the tetramethyl substituted aziridine, is a more efficient sensitizer in vitro than RSU 1069 (Ahmed et al., 1986). The extent to which variation in drug uptake can influence the sensitizing efficiency of RSU 1069 and its analogues has been investigated by determining the cellular uptake of these weakly basic sensitizers as a function of the pH of the extracellular medium (pHe) over the range 5.4-8.4. Following exposure of V79 cells to these agents for 1 h at room temperature, the ratio of intra- to extracellular concentration (Ci/Ce) was near unity at pH 5.4. Increasing pHe to 8.4 resulted in no change in the ratio Ci/Ce for RSU 1069 (pKa = 6.04). In contrast, the values of Ci/Ce increased three-fold for RSU 1165 (pKa = 7.38) and eleven-fold for RB 7040 (pKa = 8.45). Radiosensitization by RSU 1069 showed little dependence on pHe over the range studied, whereas increasing pH caused an apparent increase in sensitizing efficiency of both RSU 1165 and RB 7040. However, when the enhancement ratios for sensitization were normalized to take account of the effect of extracellular pH on drug uptake, efficiency of sensitization was independent of pHe. This study suggests that changes in basicity (pKa) may have wider potential for therapeutic exploitation on the basis of selective tumour uptake for this type of agent.     

Effects of laser photodynamic therapy on tumor phosphate levels and pH assessed by 31P-NMR spectroscopy

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.     

Low pH does not affect the dose response for 5'-amino-5'-deoxythymidine modulation of IdUrd DNA incorporation and radiosensitization in a human bladder cancer cell line.

We report that coincubation of 647V cells for one cell cycle with low concentrations (30 microM) of 5'-amino-5'-deoxythymidine increased IdUrd DNA incorporation and radiosensitivity at low extracellular pH (pHe 6.8) in a fashion similar to treatment at normal pHe. IdUrd DNA incorporation is inhibited by high (300 microM) 5'-AdThd concentrations at both normal and low pHe (7.4 and 6.8), resulting in no significant radiosensitization. These results at low pHe were not anticipated based on previously published studies of 5'-AdThd modulation of thymidine kinase (TK) activity and nucleoside cellular uptake. Our results suggest that regulation of intracellular pH (pHi) during the course of one cell cycle negates the 5'-AdThd dose-dependent modulation of TK activity demonstrated previously. Flow cytometric measurement of pHi in 647V cells showed that normal pHi (pH 7.4) was maintained in 647V cells over a 12- to 24-h exposure to low pHe (pH 6.8). Thus the concomitant use of IdUrd and high concentrations of 5'-AdThd (> 30 microM) is unlikely to result in selective in vivo radiosensitization of human tumors under conditions which are intermittently or chronically acidic. However, low concentrations of 5'-AdThd may prove to be an effective in vivo modulator of IdUrd radiosensitization of human tumors under both normal and acidic conditions.