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Quercetin has been shown to act as a hyperthermia sensitizer by inhibiting the synthesis of heat shock protein 70 (HSP70) in a variety of tumour cell lines. It is most effective under conditions of low pH. This study was designed to test the hypothesis that quercetin suppresses thermotolerance development in cells adapted to growth at low pH and renders them as responsive as acutely acidified cells to hyperthermia-induced cytotoxicity. Chinese hamster ovarian carcinoma cells (OvCa) were exposed to 42 degrees C hyperthermia and/or quercetin (50-200 mm) at their growth pH of either 7.3 or 6.7 or after acute acidification from 7.3 to 6.7. Thermotolerance development was measured by colony survival. HSP70 synthesis and total protein synthesis were measured by radioactive precursor pulse labelling techniques. Quercetin, in a concentration-dependent manner, reduced the rate of total protein synthesis and increased cytotoxicity equally after acute acidification to pH 6.7 or growth at pH 6.7 at 37 degrees C, and to a greater extent than it did in cells at pH 7.3. At 42 degrees C, 100 mm quercetin inhibited total protein synthesis, HSP70 synthesis and thermotolerance development to a similar extent in cells grown at pH 6.7 or acutely acidified to pH 6.7. In contrast, quercetin reduced but did not completely inhibit HSP70 synthesis and thermotolerance development in cells grown and heated at pH 7.3. These results support the hypothesis that quercetin can specifically reduce thermotolerance development in tumour cells adapted to growth at pHe 6.7 so that they respond similarly to acutely acidified cells. Since many tumours are adapted to growth at low pH and may resist a wide variety of therapeutic modalities, inhibition of thermotolerance expression by quercetin may not only enhance the response to hyperthermia but the response to commonly used therapies such as chemotherapy and radiation.

Melanoma cells in vivo maintain intracellular pH (pHi) in a viable range despite an extracellular tumor pH (pHe) that is typically below 7.0. In general, three families of transporters are capable of removing metabolic protons, but the specific transporters responsible for the maintenance of pHi at low pHe in melanomas have not been identified. Although the transporters exist in most cells, an inhibitor would be predicted to have selectivity for cells located in an acidic tumor bed because cells in that environment would be expected to have transporters chronically activated. In this report, the levels and extent of expression of the Na+/H+ exchanger (NHE-1) and two of the H+-linked monocarboxylate transporters (MCTs) were evaluated in three melanoma cell lines. The effects of inhibitors of each transporter were tested at an extracellular pH (pHe) of 7.3, 6.7, or 6.5 in melanoma cells that were grown at pHe 7.3 or 6.7. The activity of MCT isoform 1 (MCT-1) was up-regulated in three melanoma cell lines at low pHe, but that of NHE-1 was not. Furthermore, NHE-1 activity was lower in the melanomas than in other normal and malignant cell lines that were tested. Reverse transcription-PCR using primers specific for MCT-1, MCT-4, and NHE-1 showed that expression of none of these transporters was reproducibly up-regulated at the level of transcription when cells were grown at pHe 6.7 instead of pHe 7.3. Ex vivo experiments using DB-1 human melanoma xenografts grown in severe combined immunodeficient mice found that MCT-1 and not NHE-1 was a major determinant of DB-1 tumor cell pHi. Taken together, the data indicate that MCTs are major determinants of pH regulation in melanoma. In contrast, keratinocytes and melanocytes under low pHe conditions relied on NHE-1. Inhibitors of MCTs thus have great potential to improve the effectiveness of chemotherapeutic drugs that work best at low pHi, such as alkylating agents and platinum-containing compounds, and they should be selective for cells in an acidic tumor bed. In most tissues, it is proposed that the NHE-1 could compensate for an inhibited MCT to prevent acidification, but in melanoma cells this did not occur. Therefore, MCT inhibitors may be particularly effective against malignant melanoma.

Angiostatin inhibits angiogenesis by binding to endothelial cells (ECs) lining the vasculature of growing tumors. These cells are in a dynamic state during angiogenesis and are thus not firmly attached to the extracellular matrix. This makes them more vulnerable to anoikis, a process resulting in cell death initiated by or promoted by loss of attachment. Another potential source of EC vulnerability during tumor angiogenesis is that tumor extracellular pH is typically lower than in normal tissues. This presents an additional challenge to ECs in terms of maintaining ionic homeostasis. We report here that the lethality of angiostatin is significantly enhanced both by reduced matrix attachment during exposure and lowered extracellular pH (pH(e)). Another effect of angiostatin at reduced pH(e) is a decreased intracellular pH (pH(i)). These effects were observed in three model systems: aortic ring sprouts, ECs during tube formation, and ECs in a scratch/migration assay. In these three dynamic assays, angiostatin-induced cell death and intracellular acidification were clearly seen when pH(e) was reduced to 6.7. The intracellular acidification was far greater than that induced by pH(e) reduction alone. In contrast, the effect of angiostatin on pH(i) and on viability were not observed in a subconfluent monolayer in which the cells were allowed to attach to substrate for 48 h prior to exposure to angiostatin. These data suggest that low pH(e) and reduced adhesion to matrix play a role in the specificity of angiostatin for tumor neovasculature in contrast to wound healing and other normal angiogenic processes. The results also implicate roles for both pH(e) and pH(i) regulation in the mechanism of angiostatin action.

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification., (Copyright 2000 Wiley-Liss, Inc.)

Cells which have been adapted to growth at low extracellular pH (pHe) typically develop both an upregulation of steady state intracellular pH (pHi) and an ability to develop thermotolerance to 42 degrees C hyperthermia. These properties were acquired at different times, however. Days were required at pHe = 6.70 for two cell lines to adapt to low pHe by the thermotolerance criterion, but both had elevated steady state pHi values after only 4 hours at pHe = 6.70. A better correlation with adaptation to low pHe (as defined by hyperthermia) was found with changes in proton extrusion and the rate of pHi recovery after cytosolic acidification.

Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/ H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl-/HCO(-3)exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification.

Purpose: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi., Methods and Materials: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7., Results: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival., Conclusions: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.

A CHO cell model is described for in vitro studies of the mechanisms underlying heat resistance in cells adapted to growth in acidic environments. Adaptation is defined as a loss of pH 6.7-induced sensitization to 42.0 degrees C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pHi). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7G cells heated (42.0 degrees C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3G cells) and heated at pH 7.3, while the SF of 7.3G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7G cells to killing was observed at 42.0 degrees C, it was not observed at 43.0 and 45.0 degrees C. Both 6.7G and 7.3G cells were able to develop comparable levels of thermotolerance during 42.0 degrees C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7G cells to develop thermotolerance during heating at 42.0 degrees C. The acquired 6.7G phenotype reverted to the 7.3G phenotype following 17 days of culture at pH 7.3. Amiloride (0.5 mM), an inhibitor of the Na+/H+ exchanger (NHE), did not sensitize 7.3G and 6.7G cells to 42.0 degrees at their growth pHs. However, sensitization was observed for acutely acidified 7.3G cells. This is consistent with the hypothesis that extracellular acute acidification causes a decrease in pHi, and that the recovery from that decrease is achieved in part by activation of the NHE. An elevation of steady-state pHi, measured by analysing intracellular BCECF excitation spectra, was documented in a suspension assay for cells grown at pH 6.7 for 180 days. The elevation was bicarbonate-dependent (negligible in the absence of HCO3-, +0.17 pH units in the presence of HCO3-). These results suggest that the altered regulation of pHi in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.

HSP27 levels are elevated in two Chinese hamster cell lines and in a human melanoma cell line adapted to growth at pH 6.7. The level of HSP72 is elevated in the melanoma cell line but not in the hamster cell lines adapted to growth at pH 6.7. HSC73 levels are not elevated in any of the adapted cell lines. Low pH adapted cells from all cell lines are resistant to cisplatin. It is proposed that elevated HSP27 levels in low pH-adapted cells may play a role in resistance to hyperthermia and resistance to cisplatin.

Since low intracellular pH potentiates the hyperthermia response, the number of protons generated within the cell is calculated for both aerobic and anaerobic metabolism. The calculations incorporate the most recent estimates for the P: O ratio in mitochondrial oxidative phosphorylation, and include the hydrolysis of ATP as well as its synthesis. Anaerobic glycolysis of glucose to lactate generates two proton equivalents per mol of glucose, due to the complete dissociation of lactic acid. This corresponds to 1.00 proton per ATP, and is independent of pH. For aerobic metabolism of glucose to CO2 and water, protons are generated in the cell by dissociation of carbonic acid, and the exact number depends upon cytosolic pH. In the physiological pH range, one finds 4.1-5.9 mol of hydrogen ion per mol of glucose, which corresponds to 0.14-0.20 per ATP. Thus, anaerobic metabolism generates 5-7x as much acid within the cell as aerobic metabolism if the two are operating at the same rate in terms of ATP generation per unit time.

Amifostine, a chemo- and radioprotective agent developed as adjunctive therapy for malignancies, induces hypotension after approximately 20% of patient administrations. This study examines the molecular mechanisms underlying hypotension induced by amifostine. Amifostine and its metabolite, WR-1065, induced dose-dependent hypotension in anesthetized rats that was not blocked by N(G)-methyl L arginine (L-NAME), an NO synthase inhibitor. WR-1065 but not amifostine induced concentration-dependent relaxation of isolated rat aortic rings in an endothelium-independent fashion. Relaxation was not associated with increases in cGMP or cAMP and could not be blocked by L-NAME or indomethacin. Similarly, neither amifostine or WR-1065 activated adenylyl, particulate guanylyl, or soluble guanylyl cyclases. WR-1065 relaxed rat aortic rings precontracted with norepinepherine, suggesting alpha-adrenergic blocking activity. However, neither amifostine nor WR-1065 altered the ability of prazosin or phentolamine to bind to alpha-adrenergic receptors. Further, WR-1065 had no effect on receptor-mediated increases in intracellular calcium in BAL 17 murine B lymphocytes in vitro. Thus, hypotension after administration of amifostine is mediated by WR-1065 and appears to result from direct relaxation of vascular smooth muscle. Smooth muscle relaxation induced by WR-1065 is not related to production of nitric oxide, prostaglandins, or cyclic nucleotides; alpha-adrenergic receptor antagonism; or interference with receptor-dependent increases in intracellular calcium. Administration of ephedrine, an efficacious adrenergic agonist, attenuated hypotension induced by amifostine in anesthetized rats and may be useful in alleviating hypotension associated with amifostine administration in patients.

As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42 degrees C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42 degrees heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO3-. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells.

Fluorescent measurements of intracellular H(+) and Na(+) are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength "windows" in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na(+)]i with excellent linearity over the range 2-150 mM and with an accuracy of approximately 5 mM.

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca2+] and [Cd2+], or [Ca2+] and [Ba2+], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba2+] was added to a B cell line that had been stimulated with antiimmunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca2+], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba2+. The conductance for Ba2+ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca2+] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba2+.

Dequalinium (4) is a potent and selective blocker of small conductance Ca2+-activated K+ channels, an important but relatively little studied class. The 4-NH2 group of dequalinium has been shown to contribute significantly to blocking potency. In this study, we have investigated further the role of the 4-NH2 group. Replacement of this group by other substituents (R4) and quantitative structure-activity relationship (QSAR) analysis on the resultant analogues have yielded a correlation between blocking potency and sigma R for R4 for seven of the compounds. The application of calculated electronic indices enabled the extension of the QSAR to compounds for which the appropriate sigma R values are not available, allowing all 13 analogues of this series to be included in the correlations. Analysis using electronic indices obtained from AM1 MO calculations on model compounds revealed that the blocking potency correlates with the partial charge on the ring N atom, ELUMO, and EHOMO. The EHOMO correlation is qualitatively inconsistent as the HOMO is not the same orbital in all compounds. The ELUMO correlation [pEMR = 1.19(+/- 0.21)ELUMO + 5.41(+/- 1.05), n = 13, r = 0.86, s = 0.274] suggests that the higher the ELUMO the more potent is the analogue. This is consistent with simple charge transfer from the channel to the blocker and may refer to other processes which are important for the strength of the drug-K+ channel interaction such as the desolvation of the compounds.

Fluorescence emission and triplet-triplet absorbance spectroscopy of the single tryptophan in cod parvalbumin were used to study the stability and dynamics of the protein as influenced by Ca2+ binding and interaction with a chaotropic agent. The concentrations for half-saturation for Ca binding were 3.6 x 10(-9), 3.3 x 10(-4), 7.1 x 10(-3), and 0.14 M in the presence of 0, 2, 3, and 4 M guanidine hydrochloride, respectively. As predicted for thermodynamic reversibility, the guanidine hydrochloride unfolding reaction depends upon Ca2+, and the delta G are as follows: 22.9, 29.3, 35.2, and 44.2 kJ/mol for no added Ca2+, 1, 2, and 5 mM Ca2+, respectively. The stability toward denaturation imparted by the binding of two Ca2+ is about -60 kJ/mol. For Ca(2+)-bound parvalbumin in the presence of excess Ca2+, the decay of the triplet state tryptophan is approximately exponential, and the lifetime decreases from 6.5 to 3.8 ms as the temperature increases from 10 to 40 degrees C. In contrast, the triplet decay of the calcium-free protein is nonexponential over the time range of microseconds to milliseconds, a result that may indicate that the Ca-free protein is molten-globule-like. At Ca2+ concentrations where the protein is partially saturated with Ca2+, the lifetime of the longest decay component is less than that for the Ca-saturated protein; this finding suggests an exchange of Ca2+ and a conformational change during the triplet lifetime. From these data, a rate constant for the process that includes calcium-related protein conformational change can be surmised to range between 200 and 500 s-1.

The fluorescent chelator Indo-1 can simultaneously provide information about two ions, such as Ca2+ and Ba2+, if spectra after binding can be distinguished from each other and from the ion-free dye. In the case of extracellular Ca2+ and Ba2+, the three spectra were found to be unique and independent, in the sense that no one of them could be approximated by any combination of the other two. Upon binding Ba2+, Indo-1 experienced a peak shift from 470 to 455 nm, whereas the peak for Ca(2+)-bound Indo-1 occurred at 405 nm. The computer could distinguish a contribution from Ba2-bound Indo-1 just as well when it was in a mixture containing contributions from free dye and Ca(2+)-bound Indo-1. The program used all the spectral data in a rapid noniterative calculation. It found the unique combination of Ca(2+)-bound, Ba(2+)-bound, and metal-free Indo-1 spectra which best matched the data. The spectral weighting factors which formed the best combination spectrum then corresponded to the relative concentrations of the dye in the three states. From these and the dissociation constants for Ca2+ and Ba2+, it was possible to calculate both [Ca2+] and [Ba2+] from a single data spectrum. The method was also applied to Mn2+, a metal which does not generate a unique spectrum, but which quenches Indo-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidase-conjugated anti-surface immunoglobulin (sIg) was used quantitatively to monitor endocytosis of crosslinked sIg on murine B lymphocytes. The role of biochemical second messengers in the initiation of endocytosis was assessed by employing several inhibitors. A novel peroxidase detection system was used and temperature-dependent decreases in sIg density on immunoperoxidase-labelled murine lymphocytes were monitored. Metabolic inhibitors as well as colchicine and cytochalasin D were utilized to confirm that the internalization of sIg could be blocked by classical inhibitors of the endocytosis process. The role of tyrosine kinase activity was established by the fact that endocytosis was significantly reduced with 100 micrograms/mL genistein. Experiments using EGTA or 1,2-bis(beta-aminophenoxy)ethane-N-N,N'-tetraacetic acid (BAPTA) to chelate Ca2+ indicated that Ca2+ plays little role in endocytosis. Likewise, protein kinase C (PKC) was not found to be involved in endocytosis, as activation of PKC with 50 ng/mL phorbol 12-myristate 13-acetate, or inhibition of the enzyme with 1 nmol/L or 5 nmol/L staurosporin, did not modulate endocytosis. Taken together, results suggested that ligand-induced endocytosis of antigen receptors is mediated primarily through localized membrane events and is not dependent upon the classical B lymphocyte activation signals, such as the biochemical events in the inositol phosphate cascade.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.

Carboxy-SNARF-1 is an emission-changing, pH-sensitive probe for measurements of intracellular pH. However, the protonated and deprotonated forms of the dye interact differently with intracellular constituents, and this imposes new requirements on the calibration of the system. Whole spectra of intracellular and extracellular C.SNARF-1 were analyzed and showed (1) intracellular quenching which was significantly greater for the deprotonated form of the dye than for the protonated state and (2) a detectable change in pK a. Importantly for avoiding damage to cells, this mathematical analysis allowed reference spectra for fully protonated and fully deprotonated dye to be obtained without a need for spectra measured at extreme values of pH. It is not known what constituent(s) of the intracellular milieu might be responsible for the changes in dye behavior in the cell. To address this question, preliminary experiments with cell-free buffers compared the pattern seen inside the cell with quenching by a protein (bovine serum albumin; BSA) or that due to ethanol. The BSA result was completely unlike the intracellular case in that the protonated form of the dye was quenched. Buffer containing ethanol, on the other hand, was able to mimic the essential features of the intracellular spectra.

In the theory of measurement of calcium ion activity by determination of Fura-2 fluorescence at two excitation wavelengths, the accuracy of the result depends upon the accuracy both of the sample measurements and of the calibration measurements which are made on calcium-bound and free dye. Two factors underlie adequate calibration and accuracy. The first is the elimination of systematic error due to spectral shifts arising from the intracellular environment felt by the dye. To this end, detailed comparisons between complete spectra of both calcium-bound and calcium-free Fura-2 can be used to help separate spectral effects due to light absorption by cellular constituents versus polarity and viscosity of the intracellular milieu. The second major factor which determines accuracy is the experimental uncertainty (in both sample and calibration measurements). For samples in which the ratio of bound to free dye is large, the uncertainty in the ratio is also large, even when it is expressed as a percentage of the ratio itself. The errors in calibration measurements impact on the accuracy of the method primarily through the measurements made at wavelengths which are off the spectral peaks of the bound or free dye, since these are the least accurate. In order to obtain a guide to the choice of wavelengths and estimation of the reliability of results, a mathematical expression is derived for the dependence of the accuracy of the method on the accuracy of both sample and calibration measurements.

Several mechanisms might account for the difference between spectra of Indo-1 measured on intracellular and on extracellular dye. Experiments to discriminate between various possibilities used quantitative analyses of intracellular and extracellular Indo-1 spectra. Intracellular calcium-free dye showed a 20-nm blue shift, when compared with the extracellular case. However, the calcium-bound spectrum was unaffected by the intracellular milieu. This eliminated the possibility that the spectral shift was due to calcium-independent unhydrolyzed dye. The spectral shift was reversible and was seen in resting cells as well as calcium-depleted cells. Since the apparent dissociation constant for calcium was not detectably different inside the cell, the possibility of competitive binding to another divalent cation, such as Zn2+, was eliminated as the principle source of the spectral shift. The shift appears to be due to a noncompetitive solvent effect on the emission of the calcium-free dye that is absent in the calcium-bound form.

Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface--i.e., kq = Aexp(-r/rho), where A contains a geometrical factor dependent on tryptophan burial and surface geometry [corrected]. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electron-transfer rate on distance in a protein environment and evaluate the distance parameter, rho, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donor-acceptor pair with ket = koexp(-r/rho), ko approximately 10(9) sec-1.

The triplet state absorption and phosphorescence of Zn and Pd derivatives of myoglobin were compared. Both metal derivatives exhibit long triplet state lifetimes at room temperature, but whereas the Pd derivative showed exponential decay and an isosbestic point in the transient absorption spectra, the decay of the Zn derivative was nonsingle exponential and the transient absorption spectra showed evidence of more than one excited state species. No difference was seen in triplet quenching by oxygen for either derivative, indicating that differences in the polypeptide chain between the two derivatives are not large enough to affect oxygen penetrability. Quenching was also observed by anthraquinone sulfonate. In this case, the possibility of long-range transfer by an exchange mechanism is considered.

The luminescent properties of metal-free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivates bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal-free 'porphyrin' cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70 degrees C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.

In three model systems, particles the size of cells or smaller have been surface labeled with ferritin to make them slightly paramagnetic, by virtue of the iron in the ferritin. In each case it was possible to show that labeled particles could be magnetically removed from a flowing suspension by the high-gradient magnetic separation (HGMS) technique. The first system of particles consisted of small (1 micron) carboxylate-modified latex spheres to which ferritin was covalently bound to create stable paramagnetic particles analogous to a ferritin-labeled subcellular membrane preparation. In the second system polyacrylamide beads that more closely approximated whole cells in size (5-50 microns) were labeled with immunoferritin. The third system was a biomembrane preparation: erythrocyte ghosts labeled with a ferritin-lectin conjugate. A field of 7 T (tesla) (70 kG) was used in each case, along with buffer flow rates through the HGMS column in the range 0.1-1.0 ml/min.

Porphyrins and activities of heme biosynthetic enzymes in Taenia solium cysticerci from porcine and human hosts, were examined in order to clarify the possible step where heme synthesis is interrupted. Porphyrins in the vesicular fluid of the parasite were predominantly coproporphyrin, followed by penta-carboxylated porphyrin, which together accounted for 90% of the accumulated porphyrins. Coproporphyrin and penta-carboxylated porphyrin were both type I and III isomers. Small amounts of protoporphyrin and uroporphyrin, and trace amounts of tri-, hexa- and hepta-carboxylated porphyrins were also detected. Fluorescence and phosphorescence spectra and lifetime studies revealed that at least 75% of the porphyrins were bound to metal, probably Zn, while the rest was free. Reverse phase high performance liquid chromatography monitored at an excitation wavelength of 417 nm and at an emission wavelength of 585 nm demonstrated that approximately 90% of these porphyrins were Zn-coproporphyrin. A fluorescence excitation peak at 283 nm with an emission peak at 585 nm and 625 nm indicated that some of the porphyrins were associated with proteins in the vesicular fluid of the parasite. Low levels of delta-aminolevulinic acid dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase activities, and heme concentrations were found in the extract of the parasite walls and scolex, but not in the vesicular fluid. The porphyrin accumulation pattern in this parasite can best be explained by postulating a deficiency of coproporphyrinogen oxidase activity, similar to that in human patients with hereditary coproporphyria. A parasite dissected from a human host was considerably less porphyric than those from pigs, but the pattern of accumulated porphyrins was quite similar in both. In view of their porphyrin contents, T. solium cysticerci could be light sensitive.

The binding of biotin by avidin has proven to be a useful adjunct to antigen-antibody reactions in a number of immunological labeling systems. This work has applied it to rosette formation, for identification and separation of selected lymphocytes. Three procedures were compared which incorporated the biotin-avidin reaction into a model system for the rosetting of murine T lymphocytes. The reaction was most effective in conjunction with antibodies binding to the red cell, instead of using red cells which were biotinated on endogenous cell surface proteins. Physical separation of these rosettes from unrosetted cells was demonstrated with magnetic filtration, in a preliminary experiment. One interpretation of differences in effectiveness of rosette formation in the three systems tested is in terms of the flexibility of the antibody molecule, as compared with avidin. The principal finding was that, if employed in a multiple-stage 'sandwich' labeling system, avidinbiotin binding was fully effective and provided new versatility in the design of such rosetting schemes.

Fluorescence energy transfer experiments by steady-state and nanosecond monophoton techniques were carried out with a covalently linked hybrid rabbit IgG antibody containing one antilactose site and one anti-Dns [5-(dimethylamino)-1-naphthalenesulfonyl] site. The hybrid antibody was prepared from antilactose and anti-Dns antibody by mild reduction, dissociation into half-molecules in acid, and random reassociation with re-formation, to the extent of 80%, of the single disulfide bond between the heavy chains. Fractionation with an antilactose-specific immunoadsorbent yielded a population in which each IgG molecule contained no more than one anti-Dns site per antibody. The acceptor molecules used for intramolecular energy transfer were derivatives of p-aminophenyl beta-lactoside (PAPL): (dimethyl-amino)benzeneazo-PAPL and N-fluorescyl-PAPL. The fluorescence lifetime (24 ns) and quantum yield (0.57) of the bound Dns group were unaffected by the presence of the acceptor in the adjacent site. Three models were used to calculate the minimum distance between the adjacent sites of the IgG antibody based on the overlap in the emission and absorption spectra of the donor-acceptor pairs and the segmental flexibility of the immunoglobulin molecule. The calculations yielded values in the range of 5.5-7.0 nm for the minimum distance of separation between the antibody sites in solution and demonstrated a substantial energy barrier to the closer approach of the sites.

The high gradient magnetic separation technique has been applied to separate paramagnetic erythrocytes from a cell suspension that also contained diamagnetic cells. Paramagnetism was induced in the red blood cells by oxidizing the iron atoms in the cell hemoglobin to the ferric state (methemoglobin). Diamagnetic cells were either untreated erythrocytes, containing oxyferrohemoglobin, or leukocytes in a suspension of mouse spleen cells. Cell suspensions were passed through a column containing 40 micron diameter stainless steel wire in a high magnetic field (33 kG). The paramagnetic cells were retained on the surface of the wire while the diamagnetic cells passed through. Elution of the paramagnetic cells was accomplished by removing the column from the magnet, in effect turning off the field.

AMino acid polymer antigens have been covalently coupled to sheep erythrocytes (SRBC) and these have been used in the high gradient magnetic separation (HGMS) technique to enrich murine lymph node cells which had specific receptors for antigen. The method was rapid and provided excellent cell recovery including the fraction retained on the HGMS column. The system could be operated to enrich preformed rosettes, or as an affinity column when preloaded with antigen-coupled SRBC. A 10-fold enrichment of plaque-forming cells was observed in either mode. The HGMS system employed a conventional electromagnet and the fractionation procedure required about 30 min for a sample of up to 10(8) cells.

Magnetic labeling by a magnetite-antibody conjugate has been combined with magnetic filtration (high gradient magnetic separation) to effect a rapid and efficient separation of a selected cell population from a suspension of single cells. Samples of more than 10(8) cells could be fractionated in about 5 min with complete recovery. The system has been applied to a model system using red blood cells (sheep or chicken) and commercial antibodies against species-determined cell surface antigens. Enrichments of labeled cells by factors of up to 37-fold were observed. The approach was relatively insensitive to details in the experimental protocol and to the number of unlabeled cells which were in the sample. Thus, the method was easy to use and can readily be scaled up to handle large samples containing 10(8) labeled cells in a total of 10(11) or more. It should be useful as a pre-enrichment scheme for suspensions in which cells of interest are rare and, consequently, very inefficiently sorted by fluorescence-activated instruments.

Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths.

Magnetic filtration of labelled cells as a way of separating leucocyte subpopulations was tested with a very simple and easy filtration device, using colloidal magnetite as the labelling reagent. In order to quantitate cell enrichment, a double label (both fluorescent and magnetic) was used, under conditions which labelled less than 10% of the cells in the initial sample. Up to 20 million cells were simply passed through a small magnetic filter with a hand-held syringe. Depletion of labelled cells in the suspension that passed through was threefold, and enrichment of labelled cells in the wash of the filter after its removal from the magnet was approximately fivefold. Factors which limited the quality of separation are discussed. Other, more preliminary, experiments found enrichments of 15- to 30-fold with the same colloidal magnetite and hand-held apparatus when the cell labelling system was more selective.

Most NMR contrast agents suggested to date have been paramagnetic. These agents, which include the transition and lanthanide metal ions as well as stable organic free radicals, do not provide effective contrast at concentrations much below 1 mM. However, the use of macromolecular ferromagnetic and superparamagnetic particles provides, for the first time, an NMR relaxation agent that is effective at subnanomolar concentrations. Two different sized superparamagnetic particles have been coupled to monoclonal antibodies with high affinity for a neuroblastoma-specific cell surface antigen. The specific binding of these particles, both in vivo and in vitro is demonstrated and the consequences for immunospecific NMR contrast are discussed.

A novel murine B lymphoma expressing membrane-associated IgA was isolated and used to compare mechanisms of signal transduction by sIgM and sIgA. Like other isotypes so far studied, crosslinking of sIgA by anti-immunoglobulin antibodies stimulates hydrolysis of inositol phospholipids and causes elevation of intracellular free calcium. Furthermore, signals generated through sIgA are coupled to elevation of c-fos proto-oncogene expression. Coupling appears to be through the protein kinase C rather than through the Ca2+ component of sIg signalling as phorbol diester, but the Ca2+ ionophore cannot mediate this effect. Thus these results, coupled with those from earlier studies, show that early signal transduction through surface immunoglobulin appears to be similar regardless of the particular isotype involved in binding ligand.

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (less than 2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20-30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20-50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90-100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80-90%.

Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-1-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo]aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p[[p-dimethylamino)phenyl]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently liked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (approximately 60%) the separation was 50-60 A. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.

Because of their long T2 relaxation time (400 ms), larvae of the tapeworm Taenia crassiceps can be distinctly imaged in the mouse peritoneal cavity by NMR. An echo time of 400 ms sharply contrasted the parasites against the surrounding abdominal organs. A rapid, quantitative assay for parasite load can be obtained by recording the amplitude of a spin echo with no applied gradients at an echo time of 400-1000 ms. The T2 of the vesicular fluid of the human parasite Taenia solium was also long (600 ms), indicating that the same approach (long TE and long TR) can be used to image this parasite for diagnosis of human cysticercosis by NMR imaging.

A receptor surface Ig (sIg) signaling variant of WEHI-231 was constructed to investigate components and linkages between various signaling events associated with signal transduction through sIg. Unlike the wildtype, crosslinking of sIgM on VS2.12-cl.2 did not result in downregulation of proliferation. Similarly, receptor crosslinking was uncoupled from inositol phospholipid (PI) hydrolysis and upregulation of c-fos expression in the variant. The signaling defect in VS2.12-cl.2 appears to be proximal to phospholipase C activation as direct G protein activation by A1F4- triggers PI hydrolysis and bypassing PI hydrolysis using phorbol diester stimulation of protein kinase C restores the inhibitable phenotype and the ability to upregulate c-fos. Even more interesting, sIg-linked Ca2+ responses by VS2.12-cl.2 are equivalent to these observed in the wildtype WEHI-231. These latter results suggest that contrary to current thought, sIg-generated signals may not be coupled to Ca2+ fluxes entirely via inositol phospholipid hydrolysis. Thus, VS2.12-cl.2 is a new and powerful tool with which to analyze signaling through sIg at the molecular level.