Antitumor activity of nigericin and 5-(N-ethyl-N-isopropyl)amiloride: an approach to therapy based on cellular acidification and the inhibition of regulation of intracellular pH

The extracellular pH (pHe) in solid tumors is frequently lower than the pHe in normal tissues, but the intracellular pH (pHi) is regulated to physiological levels. Cell killing can be achieved in an acidic environment in tissue culture by nigericin, which acidifies cells by transporting H+ from the extracellular space into the cytoplasm; this cell killing can be enhanced when used with 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of membrane-based Na+/H+ exchange, which plays a major role in the regulation of pHi (R. P. Maidorn; E. J. Cragoe; I. F. Tannock, Br. J. Cancer 67:297-303; 1993). We have therefore assessed the ability of nigericin and EIPA to kill cells in two murine solid tumors (the KHT fibrosarcoma and the EMT-6 sarcoma). Hydralazine, which reduces tumor blood flow, or glucose, which stimulates glycolysis leading to accumulation of lactate, were also administered to mice to lower pHe in the tumors. We observed only a small decrease in the surviving fractions of cells in the tumors when tolerated doses of nigericin and EIPA were given IP to tumor-bearing mice. When nigericin and EIPA were combined with administration of hydralazine, the surviving fraction of cells in both tumors was reduced by a factor of 0.01, but there were minimal effects on growth delay. Administration of glucose with nigericin and EIPA led to a smaller reduction in surviving fraction of the KHT tumor (by approximately 0.1), although glucose was more effective than hydralazine in lowering the mean tumor pHe. When KHT tumors were treated with 15 Gy X-rays followed immediately by nigericin, EIPA, and hydralazine, a reduced surviving fraction as well as an increase in tumor growth delay was observed compared to radiation alone; however, there was little evidence to suggest that these agents were selectively toxic to the cells that survived radiation. Nigericin and EIPA, with or without hydralazine, had minimal effects on normal tissues, as assessed by changes in body weight, number of leukocytes, and serum creatinine levels. We conclude that pharmacological effects to acidify cells and to prevent regulation of pHi under the acidic conditions that exist in solid tumors can lead to moderate levels of cell killing, if additional strategies are used to lower tumor pHe.     

Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH

The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro. Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways. However, their ability to buffer intracellular pH (pHi) has not been investigated. Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo. pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo. However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage. Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo. The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards. In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids. This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi. This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo. Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology. Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.     

Effects of aging on the regulation of intracellular pH in the rat jejunum

Jejunal villus cells from young-adult (6 months) and senescent (24 months) male Wistar rats were studied to evaluate the effect of aging on intracellular pH (pHi) regulation. pHi was measured by quantitative fluorescence microscopy by using BCECF-AM [2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein acetoxy methylester] under basal conditions and after inducing cytoplasmic acidification with pulsed NH4Cl. In the senescent rats, the recovery rate from the acidified levels was significantly lower than that in the young-adult rats (.208 +/- .005 vs .255 +/- .004 pH units/min). The relationship between pHi recovery and external Na+ concentration followed Michaelis-Menten type kinetics, the maximum velocity (Vmax) of alkalinization being significantly lower in the senescent rats than in the young-adult rats (.227 +/- .033 vs .297 +/- .024 pH units/min). These results indicate that the recovery of pHi from an acidic level was slower in the senescent rats, due to the reduced activity of Na+/H+ exchange as revealed by the decreased Vmax value.     

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.     

Role of putrescine in the antiporter mechanism of potassium transport in Escherichia coli and its role in the regulation of intracellular pH

The interrelation between the Escherichia coli transport processes of K+ and putrescine+2 was investigated under the inhibition and limitation of the main energy consuming potassium transport systems (Trk, Kdp). It was shown that the potassium addition to the potassium deprived putrescine loaded cells triggers the putrescine exit at the exchange on the potassium. The stoichiometry of putrescine +2/2K+ translocation provide the electroneutrality of this process. The putrescine gradient turnover changes the potassium transport direction on the opposite one. The submitted experimental data are the evidence of putrescine +2/2K+ antiporter existence. The role of this antiporter in the E. coli pH-homeostasis is discussed.     

Active oxygen generation as a possible mechanism of selenium toxicity

Selenium plays an important role in scavenging active oxygen (AO) species as an essential constituent of glutathione peroxidase. On the other hand, several reports proposed a possible induction of toxic AO by selenium compounds in vitro. However, some of these experiments including ours, were revealed to conclude on the basis of experimental artifacts, and to have problems in the interpretation of data. Methods or principles so far used for the detection of AO species generated by selenium compound were measurement of chemiluminescence from lucigenin or luminol by AO species, the spectrophotometric analysis of reduction of ferricytochrome c or nitroblue tetrazolium (NBT) by superoxide anion (O2-), electron spin resonance (ESR) spectra using dimethylpyrroline oxide (DMPO) as a spin trapping agent, the deoxyribose decomposition by hydroxyl radical (HO.), the salicylate hydroxylation by HO., and the strand breakage of DNA by AO. Many of these methods together with their principles seem to have some defects which prevent clear conclusion as stated below. (i) Lucigenin was found to mediate the formation of O2- in the presence of selenite and reduced glutathione (GSH). Therefore, lucigenin is not a suitable reagent. (ii) Luminol may also mediate O2- generation in the presence of HO.. (iii) ferricytochrome c can be reduced to ferrocytochrome c in the mixture of selenite and GSH in the absence of oxygen. Moreover, the spectrophotometric method is interfered by turbidity of elemental selenium formed under some conditions in the reaction mixture containing selenite and GSH. (iv) NBT is also reduced by selenium compounds in the absence of O2. (v) ESR signals of AO species were obtained in the reaction mixture containing selenite and GSH, or in the solution of hydrogen selenide in the presence of O2. However, selenide decomposed spin adduct of DMPO with HO. (DMPO-OH). Therefore, the intensity of the signals is not quantitative. (vi) CuZn-SOD is not necessarily a good tool to prove the involvement of O2- because it enhanced HO. generation in the reaction mixture containing selenite and GSH. Thus, we would like to emphasize that carefully designed experiments are required to further identify the molecular species of active oxygen induced by selenium compounds.     

Microvasculature of crotaline snake pit organs: possible function as a heat exchange mechanism

BACKGROUND: Lasting cognitive dysfunction throughout remission has been regarded as a biological vulnerability in schizophrenia, which may produce psychotic relapses with characteristic symptoms. Our hypothesis was that an abnormality in event-related potentials (ERPs) may be a neurophysiological marker of vulnerability to psychotic relapse in remitted schizophrenia. We conducted a 2-year follow-up study after evaluating ERP abnormalities to find a new ERP marker for schizophrenic relapse. METHODS: Visual ERPs were recorded from outpatients with remitted schizophrenia under maintenance pharmacotherapy (n = 44) and normal controls (n = 20) during a letter discrimination task. Based on the prospective study, the patients were divided into a relapse group (n = 20) and a nonrelapse group (n = 24). ERP findings that related to psychotic relapse within 2 years were analyzed. RESULTS: Compared with controls, the relapsers showed ERP abnormalities in the NA, N2, and P3 components, and the nonrelapsers in the P3 component. The peak latency of the NA potential was delayed significantly in the relapse group relative to the nonrelapse group, and predicted a psychotic relapse with about 90% probability. CONCLUSIONS: The delayed NA, which reflects early perceptual disorganization, may be a promising neurophysiological predictor of psychotic relapse in remitted schizophrenia under maintenance pharmacotherapy.     

Is the intracellular pH threshold an anaerobic threshold from the view point of intracellular events?: a brief review

Intracellular biochemical changes could be monitored noninvasivery and continuously by using nuclear magnetic resonance (NMR). In about the last decade, intracellular behavior of phosphorus compounds and pH during muscle contraction in man have been studied by 31P nuclear magnetic resonance spectroscopy (31P-MRS). During incremental load, lactic acidosis was followed by a decline in intracellular pH. 31P-MRS studies has been definitely proved that this change in intracellular pH shows the threshold behavior. Some reports discussed the intracellular pH threshold (pHT) as an anaerobic threshold (AT) from the view point of intracellular events. However, our studies revealed that pHT did not reflect the onset of lactate production. In this article, studies of intracellular pH of working muscle were reviewed in relation to an anaerobic threshold.     

Level of redox potential as a possible contributing influence in the pathogenicity of oral anaerobes

Dental plaque anaerobes may be associated with the etiology of periodontal disease. This has created an interest in the potential pathogenicity of oral anaerobes. We compared the metabolic activity of anaerobic corynebacteria (C. parvum, C. anaerobium) and corresponding aerobic species (C. diphtheriae, C. xerosis). The anaerobes exhibited lower levels of RNA synthesis, ranging from 5 to 10 fold over the aerobes. We further examined these anaerobes, plus Actinomyces naeslundi N16 (isolated from the anaerobic region of periodontally-diseased tissues), for the influence of redox potential on RNA level and antigenic function. Notable increases in RNA were found at specific Eh levels; the extent and direction of the changes varied with the different organisms. This environmental feature appeared to effect corresponding changes in agglutinability and PCA reactivity with antisera against the anaerobes cultured at different redox potentials. For example, while antisera against certain organisms (C. parvum, A. naeslundi) cultured under the most reuced conditions showed an intense PCA reaction, other antisera against the same organism cultured under less reduced conditions were non-reactive. Hence, alterations in redox potential may lead to alteredetabolism and to altered antigencity. Our results imply such a microbial response to environmental stress.     

Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases

The electronic absorption spectrum, susceptibility to fluoride inhibition, redox potential, and substrate turnover of several fungal laccases have been explored as a function of pH. The laccases showed a single spectrally detectable acid-base transition at pH 6-9 and a fluoride inhibition that diminished by increased pH (indicating a competition with hydroxide inhibition). Relatively small changes in the redox potentials (< or = 0.1 V) of laccase were observed over the pH 2.7-11. Under the catalysis of laccase, the apparent oxidation rates (kcat and kcat/Km) of two nonphenolic substrates, potassium ferrocyanide and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), decreased monotonically as the pH increased. In contrast, the apparent oxidation rates (kcat and kcat/Km) of three 2,6-dimethoxyphenols (whose pKa values range from 7.0 to 8.7) exhibited bell-shaped pH profiles whose maxima were distinct for each laccase but independent of the substrate. By correlating these pH dependences, it is proposed that the balance of two opposing effects, one generated by the redox potential difference between a reducing substrate and the type 1 copper of laccase (which correlates to the electron transfer rate and is favored for a phenolic substrate by higher pH) and another generated by the binding of a hydroxide anion to the type 2/type 3 coppers of laccase (which inhibits the activity at higher pH), contributes to the pH activity profile of the fungal laccases.     

Long chain analogs of physostigmine as potential drugs for Alzheimer''s disease: new insights into the mechanism of action in the inhibition of acetylcholinesterase

A model of the rabbit sinoatrial action potential is introduced, based on a model by Morris & Lecar. One cell is described by two nonlinear first-order ordinary differential equations, with ten constant parameters. The model is much simpler than most other models in use, but can reproduce perfectly experimentally recorded action potentials. The dynamics of two coupled cells, with and without the presence of periodic acetylcholine pulses, shows examples of bifurcations and strange attractors, mathematical phenomena characterizing chaotic motion. It remains to be clarified whether such dynamics is actually observed, for example in the small irregular variations of the normal heart rate.     

Accelerated repopulation during fractionated irradiation of a murine ovarian carcinoma: downregulation of apoptosis as a possible mechanism

PURPOSE: To test whether accelerated tumor clonogen repopulation occurs during continuous fractionated radiotherapy of a slow-growing mouse ovarian tumor, and if so whether the accelerated rate of repopulation is predicted by the pretreatment potential doubling time, and whether changes in apoptotic response are a possible mechanism for this change. METHODS AND MATERIALS: The rate of clonogen production during fractionated radiotherapy was followed using the tumor-control assay, with an independent determination of the sensitivity to repeated dose fractions in vivo in the absence of repopulation. The pretreatment potential doubling time was measured by bromodeoxyuridine (BrdUrd) labeling and fluorescence measurements. The apoptotic and mitotic indices at various times during treatment were scored histologically. RESULTS: The slow-growing (pretreatment volume doubling time 6 days) ovarian tumor OCA responds to daily irradiation with 6 Gy under hypoxia by negligible tumor clonogen production in the first few days, followed by a change at about 9 days to accelerated repopulation, after which the effective clonogen doubling time Tclon was about 2 days, near the pretreatment Tpot of 1.7 days. Alternative interpretations of the data, such as a change in radiosensitivity vs. a change in the repopulation rate or acceleration at 3 days as opposed to 9 days, were shown to be unlikely. This change was accompanied by a reduced apoptotic response (measured morphometrically). CONCLUSIONS: When sensitivity to fractionated doses has been corrected for in vivo, this slow-growing mouse tumor exhibits a change to accelerated clonogen production during a continuous radiotherapy regimen that is accompanied or preceded by a reduced histologic apoptotic response. Tclon during accelerated repopulation was slightly longer than the pretreatment Tpot.     

Extracorporeal photochemotherapy (photopheresis) induces apoptosis in lymphocytes: a possible mechanism of action of PUVA therapy

The mechanism of action of psoralen plus UVA (PUVA) and photopheresis is not entirely understood. These therapies are assumed to be immunomodulating partly by gradually decreasing leukocyte viability. We investigated whether this delayed form of cell death was due to apoptosis. Untreated and treated (PUVA exposed) leukocytes obtained from six patients with systemic sclerosis and (untreated) leukocytes from healthy control individuals were studied. Qualitative gel electrophoresis and quantitative in situ nick translation analysis of DNA fragmentation was performed. Apoptosis of the treated cells did occur (gel electrophoresis) after 24 h. At t = 0 h, immediately after exposure to PUVA, there was no evidence of DNA fragmentation in the treated cells. The percentage of treated cells undergoing apoptosis was 20-55% at t = 24 h (in situ nick translation). The untreated leukocytes of the patients and the healthy individuals showed no distinctive rise in apoptotic cells. Apoptosis of the leukocytes after PUVA or photopheresis treatment might be a mechanism of action and might explain the therapeutic response.     

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.     

Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis

Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.     

Habituation of the orienting response: a gating mechanism subserving selective attention

Possibilities of involution of changes in lesser circulation after closure of experimental aortopulmonary anastomosis were studied. 37 observations at various intervals after closure of anastomosis (several minutes to 13.5 months) in 25 dogs were analyzed. Before closure the anastomosis had functioned for 1-7 months. The results of histological examinations of lungs, pressure measurements in lesser circulation, heart weight, electrocardiographic and spirographic examinations were analyzed. It was found that complete involution of changes in lesser circulation was possible only in first month of existence of anastomosis, in this case with changes of both "early" and "late" types. "Late"-type changes after four months function of anastomosis had both reversible and irreversible character, whereas "early"-type changes became irreversible already after three-month duration of anastomosis. With the "late"-type changes, the operation itself (closure of anastomosis) was accompanied by symptoms of pulmonary vasomotor paresis and heart failure, whereas in the presence of "early"-type changes the operation elicited no morphological or functional changes.     

Autocatalytic oxidation of hemoglobin by nitrite: a possible mechanism

Oxidation of oxyhemoglobin by nitrite ions to produce methemoglobin is one of the more employed procedures to oxidize the hemoprotein. The process takes place readily after a clear induction time. This behaviour is usually explained in terms of an autocatalytic reaction mechanism. However, the generally accepted mechanism is not autocatalytic and cannot explain the main features of the process. In the present work it is proposed that the characteristics of the process require the occurrence of a fast reaction between oxyhemoglobin and nitrogen dioxide. This process acts as a branching step, leading to the observed autocatalysis.