Dynamic pH Changes Research Articles

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH.

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.

A new slurry pH model accounting for effects of ammonia and carbon dioxide volatilization on solution speciation

Ammonia (NH3) emissions from manure constitute a significant loss of fixed nitrogen (N) from agricultural systems and contribute to air pollution and ecosystem degradation. Accurate models of such NH3 emissions will improve our understanding of the factors that control the emissions and allow appropriate mitigation actions to be identified and quantified. Although the importance of manure pH on ammonia emission has been recognized for decades, the physical and chemical interactions that control pH are not fully understood. Here we present a novel mathematical model that includes the dynamic and crucial pH changes in the surface of stored slurry or slurry applied in the field. In the model, slurry pH is calculated by simultaneously determining: (1) speciation of the acid–base reactions, (2) diffusion of each buffer species, and (3) emission of NH3 and CO2. New features of the model include a reduced variable that combines time and location and an analytical approach to solving the resulting system of equations using Mathematica. To evaluate the model, we made measurements of pH at a resolution of 0.1 mm in the top 30 mm of an ammonium bicarbonate solution. These measurements show the creation of a large pH gradient (>1 pH unit in <30 mm after 20 h) and its change over time due to simultaneous NH3 and CO2 emission from aqueous solutions. The model was able to accurately predict the development of pH gradients over time, suggesting that our understanding of the factors controlling pH is correct. New developments presented in the model should be useful for future work on understanding and predicting NH3 emission from manure.

Bioactivity Behavior of YSZ-Al&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;/10HAP Bioceramics Composites in Simulated Body Fluid

Yttria-stabilized zirconia and alumina made significant contributions to the development of health care industry, specifically as orthopedic and dental materials. Both bioceramics are nearly inert ceramics, as they do not allow the interfacial bonding with tissue. Thus, it is necessary to provide bioactive surrounding as to elicit a specific biological response at the interface of material. This research reported the microstructure and bioactivity behavior of YSZ-Al2O3/10HAP with 30 wt. % and 60 wt. % of YSZ content. Powders were mixed before being compacted at 225MPa using uni-axial press machine. The composites were sintered at 1200 ̊C with heating rate of 10 ̊C/min. In-vitro bioactivity behavior of the composites were evaluated by immersing the composites into simulated body fluid. Results from x-ray diffraction pattern, confirmed the phase formation of apatite by the presence of Ca2P2O7, and CaO that might be useful on implant cell interaction in a body environment. The apatite formation was observed on the surfaces of the composites by SEM only after 9 days of immersion and subsequently apatite nucleation increased with prolonging immersion time. The dynamic changes in pH, between ion concentration in SBF and bioceramics surfaces correspondedwith an immersion time. Up to 30 days of immersion, the pH value of SBF stabilized approximately around pH 7.4-7.6, similar to the human blood plasma. Formation of apatite on composites surface of prepared YSZ-Al2O3/10HAP bioceramics may contribute to the improved biocompatibility and osteoconductivity.

Assessing the Risk of pH-Dependent Absorption for New Molecular Entities: A Novel in Vitro Dissolution Test, Physicochemical Analysis, and Risk Assessment Strategy

Weak base therapeutic agents can show reduced absorption or large pharmacokinetic variability when coadministered with pH-modifying agents, or in achlorhydria disease states, due to reduced dissolution rate and/or solubility at high gastric pH. This is often referred to as pH-effect. The goal of this study was to understand why some drugs exhibit a stronger pH-effect than others. To study this, an API-sparing, two-stage, in vitro microdissolution test was developed to generate drug dissolution, supersaturation, and precipitation kinetic data under conditions that mimic the dynamic pH changes in the gastrointestinal tract. In vitro dissolution was assessed for a chemically diverse set of compounds under high pH and low pH, analogous to elevated and normal gastric pH conditions observed in pH-modifier cotreated and untreated subjects, respectively. Represented as a ratio between the conditions, the in vitro pH-effect correlated linearly with clinical pH-effect based on the Cmax ratio and in a non-linear relationship based on AUC ratio. Additionally, several in silico approaches that use the in vitro dissolution data were found to be reasonably predictive of the clinical pH-effect. To explore the hypothesis that physicochemical properties are predictors of clinical pH-effect, statistical correlation analyses were conducted using linear sequential feature selection and partial least-squares regression. Physicochemical parameters did not show statistically significant linear correlations to clinical pH-effect for this data set, which highlights the complexity and poorly understood nature of the interplay between parameters. Finally, a strategy is proposed for implementation early in clinical development, to systematically assess the risk of clinical pH-effect for new molecular entities that integrates physicochemical analysis and in vitro, in vivo and in silico methods.

Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

Dietary proteins extend the survival of Salmonella Dublin in a gastric acid environment.

The pH of the human stomach is dynamic and changes over time, depending on the composition of the food ingested and a number of host-related factors such as age. To evaluate the number of bacteria surviving the gastric acid barrier, we have developed a simple gastric acid model, in which we mimicked the dynamic pH changes in the human stomach. In the present study, model gastric fluid was set up to imitate pH dynamics in the stomachs of young and elderly people after ingestion of a standard meal. To model a serious foodborne pathogen, we followed the survival of Salmonella enterica serotype Dublin, and found that the addition of proteins such as pepsin, ovalbumin, and blended turkey meat to the simple gastric acid model significantly delayed pathogen inactivation compared with the control, for which no proteins were added. In contrast, no delay in inactivation was observed in the presence of bovine serum albumin, indicating that protection could be protein specific. The simple gastric acid model was validated against a more laborious and complex fermenter model, and similar survival of Salmonella Dublin was observed in both models. Our gastric acid model allowed us to evaluate the influence of food components on survival of pathogens under gastric conditions, and the model could contribute to a broader understanding of the impact of specific food components on the inactivation of pathogens during gastric passage.

Assessment of polyelectrolyte coating stability under dynamic buffer conditions in CE

Dynamic buffer conditions are present in many electrophoretically driven separations. Polyelectrolyte multilayer coatings have been employed in CE because of their chemical and physical stability as well as their ease of application. The goal of this study is to measure the effect of dynamic changes in buffer pH on flow using a real-time method for measuring EOF. Polyelectrolyte multilayers (PEMs) were composed of pairs of strong or completely ionized polyelectrolytes including poly(diallyldimethylammonium) chloride and poly(styrene sulfonate) and weak or ionizable polyelectrolytes including poly(allylamine) and poly(methacrylic acid). Polyelectrolyte multilayers of varying thicknesses (3, 4, 7, 8, 15, or 16 layers) were also studied. While the magnitude of the EOF was monitored every 2 s, the buffer pH was exchanged from a relatively basic pH (7.1) to increasingly acidic pHs (6.6, 6.1, 5.5, and 5.1). Strong polyelectrolytes responded minimally to changes in buffer pH (<1%), whereas substantial (>10%) and sometimes irreversible changes were measured with weak polyelectrolytes. Thicker coatings resulted in a similar magnitude of response but were more likely to degrade in response to buffer pH changes. The most stable coatings were formed from thinner layers of strong polyelectrolytes.

Effects of cyclic changes in pH and salinity on metals release from sediments

The effects of dynamic changes in pH and salinity on metal speciation and release are investigated with sediments posed in a simulated estuarine environment. The release of Zn, Cd, Mn, and Fe was studied using sediment from the Anacostia River (Washington, DC, USA) spiked with freshly precipitated amorphous cadmium sulfide to increase Cd content. The sediment was exposed to salt water (high pH, ionic strength) and freshwater (neutral pH, minimal ionic strength) continuously and alternately (to mimic tidal changes) in small microcosms over 100 d. At the conclusion of the experiments, the vertical profiles of acid volatile sulfide (AVS) and simultaneously extracted metals (SEM) as well as porewater metals and anion concentrations were characterized. Acid volatile sulfide oxidation at the sediment surface led to a commensurate increase in dissolved metal species and metal release that was strongly dependent on the changes in the overlying water characteristics. Total Cd release was substantially higher during exposure to salt water, although, as a result of complexation, predicted dissolved Cd(2+) concentration in the overlying water was higher during exposure to freshwater. Total Zn release was little changed during exposure to salt water and freshwater, although the predicted dissolved Zn(2+) concentration was much higher during freshwater exposures. No significant iron was released because of the rapid oxidation of ferrous iron (Fe(2+)) in aerobic surficial sediments and overlying water. The present study suggests that cyclic changes in pH and salinity in the overlying water can dramatically influence metal release from estuarine sediments.

Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2.

Climate change scenarios predict a doubling of the atmospheric CO2 concentration by the end of this century. Yet, how rising CO2 will affect the species composition of aquatic microbial communities is still largely an open question. In this study, we develop a resource competition model to investigate competition for dissolved inorganic carbon in dense algal blooms. The model predicts how dynamic changes in carbon chemistry, pH and light conditions during bloom development feed back on competing phytoplankton species. We test the model predictions in chemostat experiments with monocultures and mixtures of a toxic and non-toxic strain of the freshwater cyanobacterium Microcystis aeruginosa. The toxic strain was able to reduce dissolved CO2 to lower concentrations than the non-toxic strain, and became dominant in competition at low CO2 levels. Conversely, the non-toxic strain could grow at lower light levels, and became dominant in competition at high CO2 levels but low light availability. The model captured the observed reversal in competitive dominance, and was quantitatively in good agreement with the results of the competition experiments. To assess whether microcystins might have a role in this reversal of competitive dominance, we performed further competition experiments with the wild-type strain M. aeruginosa PCC 7806 and its mcyB mutant impaired in microcystin production. The microcystin-producing wild type had a strong selective advantage at low CO2 levels but not at high CO2 levels. Our results thus demonstrate both in theory and experiment that rising CO2 levels can alter the community composition and toxicity of harmful algal blooms.

In vitro dissolution of calcium phosphate-mullite composite in simulated body fluid

In our recent research, we have developed novel CaP-mullite composites for bone implant applications. In order to realize such applications, the in vitro dissolution behaviour of these materials needs to be evaluated. In this perspective, the present paper reports the dissolution behavior of pure hydroxyapatite (HAp) and hydroxyapatite composites with 20-30 wt% mullite in simulated body fluid (SBF). The in vitro dissolution experiments were carried out for different time duration starting from 7 days up to 28 days. XRD and SEM results show almost no dissolution for pure HAp and HAp composite with 30 wt% mullite. However, HAp-20 wt% mullite composite exhibits considerable dissolution after 7 days. The alpha-TCP phase mainly contributes to the dissolution process. Based on the dynamic changes in pH, ionic conductivity, Ca and P ion concentration in SBF as well as microstructural observations of the bioceramic surfaces after various time frames of immersion in SBF, the differences in dissolution behaviour are discussed.

Characterization of PEBBLEs as a Tool for Real‐Time Measurement of Dictyostelium discoideum Endosomal pH

The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding) PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum). Oregon Green 514‐CdSe Quantum Dot PEBBLEs were used to measure real‐time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

Tumor-associated Carbonic Anhydrase 9 Spatially Coordinates Intracellular pH in Three-dimensional Multicellular Growths

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO(2) hydration. Expression is greatly enhanced in many tumors, particularly in aggressive carcinomas. The functional role of CA9 in tumors is not well established. Here we show that CA9, when expressed heterologously in cultured spheroids (0.5-mm diameter, ~25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a near-uniform intracellular pH (pH(i)) throughout the structure. Dynamic pH(i) changes during displacements of superfusate CO(2) concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (~0.25 pH(i) reduction) and significant time delays (~9 s) for pH(i) changes in core versus peripheral regions. pH(i) non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pH(i) regulation is unaffected by CA9 expression. The influence of CA9 on pH(i) is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pH(i) spatially by facilitating CO(2) diffusion in the unstirred extracellular space of the spheroid. We suggest that pH(i) coordination may favor survival and growth of a tumor. By disrupting spatial pH(i) control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors.

Dynamic Dissolution Testing To Establish In Vitro/In Vivo Correlations for Montelukast Sodium, a Poorly Soluble Drug

The objectives of the study was to develop a dissolution test method that can be used to predict the oral absorption of montelukast sodium, and to establish an in vitro/in vivo correlation (IVIVC) using computer simulations. Drug solubility was measured in different media. The dissolution behaviour of montelukast sodium 10 mg film coated tablets was studied using the flow-through cell dissolution method following a dynamic pH change protocol, as well as in the USP Apparatus 2. Computer simulations were performed using GastroPlus. Biorelevant dissolution media (BDM) prepared using bile salts and lecithin in buffers was used as the dissolution media, as well as the USP simulated intestinal fluid (SIF) pH 6.8 and blank FaSSIF pH 6.5. Dissolution tests in the USP Apparatus 2 were performed under a constant pH condition, while the pH range used in the flow through cells was pH 2.0 to 7.5. The in vitro data were used as input functions into GastroPlus to simulate the in vivo profiles of the drug. The solubility of montelukast sodium was low at low pH, but increased as the pH was increased. There was no significant difference in solubility in the pH range of 5.0 to 7.5 in blank buffers, but the drug solubility was higher in biorelevant media compared with the corresponding blank buffers at the same pH. Using the flow through cells, the dissolution rate was fast in simulated gastric fluid containing 0.1% SLS. The dissolution rate slowed down when the medium was changed to FaSSIF pH 6.5 and increased when the medium was changed to FaSSIF medium at pH 7.5. In the USP Apparatus 2, better dissolution was observed in FaSSIF compared with the USP buffers and blank FaSSIF with similar pH values. Dissolution was incomplete with less than 10% of the drug dissolved in the USP-SIF, and was practically non existent in blank FaSSIF pH 6.5. The in vitro results of the dynamic dissolution test were able to predict the clinical data from a bioavailability study best. Dynamic dissolution testing using the flow through cell seems to be a powerful tool to establish in vitro/in vivo correlations for poorly soluble drugs as input function into GastroPlus.

Model discrimination for the propionic acid diffusion into hydrogel beads using lifetime confocal laser scanning microscopy

Hydrogels are increasingly used for the entrapment of biocatalysts, especially for the use of enzymes in organic solvent systems. Hence, it is necessary to understand the influence of the hydrogel matrix on the transport kinetics (diffusion and phase transfer) and on the reaction kinetics (activity and stability of the enzymes). Here, the diffusion of propionic acid into Ca-alginate hydrogel beads is studied using a structured methodology, model-based experimental analysis (MEXA). The basis for all further investigations is a rigorous model of all processes occurring during the diffusion of the propionic acid into the Ca-alginate hydrogel bead. As competing model assumptions the Fickian and Nernst–Planck diffusion laws are integrated. Then, an optimal experimental design is performed to determine those experiments that allow an efficient discrimination of the rival model candidates. The change in pH-value over time resulting from the diffusion of propionic acid into the hydrogel bead is observed in the centre of the hydrogel bead. The fluorescent pH-indicator resorufin shows pH-dependent lifetimes that are measured by time-correlated single photon counting using a confocal laser scanning microscope at high temporal resolution (every 1 s). To our knowledge lifetime confocal laser scanning microscopy is used for the first time in this study for the quantification of dynamic pH-changes in macroscopically large particles. The model parameters for each candidate model are estimated based on 16 independent experimental data sets. Finally, the likelihood function is used to evaluate and discriminate the competing diffusion laws. For the diffusion of propionic acid into spherical Ca-alginate hydrogel beads, it could be shown that Fickian diffusion is not able to describe the process accurately in contrast to Nernst–Planck diffusion. The influence of the Ca-alginate hydrogel density was found to be insignificant for the estimated diffusion coefficient.

Visual Microplate Determination of Hydrogen Peroxide by Autocatalytic Reaction with Sulfite and Hydrogen Peroxide

The oxidation reaction from sulfite ion to sulfate ion with hydrogen peroxide is an autocatalytic reaction, in which a proton (H+) acts not only as the reaction product, but also as the reactant. This reaction provides the characteristic S-shape pH vs. time curve, which contains the inflection time and the dynamic pH change at the end point. The threshold of the concentration of hydrogen peroxide exists in the presence of the progress of this reaction, and the value of the threshold is determined by the ratio of the concentration for several concentrations of a sulfite ion. Furthermore, the pH change at the end point can be visualized using a pH indicator, such as bromothymol blue. Based on these findings, using this reaction for the microplate, the visual determination of hydrogen peroxide by counting the discoloring color number has been developed. By the proposed method, 2.5×10−3∼1.0 mol dm−3 of hydrogen peroxide could be visibly determined. Moreover, the influence of the temperature was examined. As a result, the analytical method was able to be used within a wide range of temperatures.

Nonlinear scaling analysis of glucose metabolism in normal and cancer cells

Cancer progression is commonly accompanied by an altered glucose metabolism. In general, spatially resolved imaging of glucose metabolism and its subtle alterations might provide valuable diagnostic information in vivo. A classical example is positron emission tomography that exploits this feature in obtaining preferential accumulation of fluorescent analog of glucose in tumors, thereby achieving an imaging contrast. We report a novel scaling analysis of glucose metabolism in mammary epithelial (NMuMG) cells by detrended fluctuation analysis of Cerulean (cyan fluorescent protein variant) fluorescence. Fluorescence fluctuations of Cerulean are reasoned to be indicative of dynamic pH changes associated with glucose metabolism. Normal parental cells and the spontaneously transformed (cancerous) NMuMG cells displayed robust scaling exponent that reflects nonrandom fluctuations in Cerulean fluorescence. Acute dependence of cancer cells on glycolysis as compared with normal cells is exploited to yield a statistically significant difference in scaling exponent, thereby providing discrimination between normal and cancer cells in vitro. By careful design of experiments in vivo, the proposed scaling approach might even have diagnostic potential for early detection of cancer lesions in small animal models.

Extracellular pH and stimulated neurons.

The use of ion-sensitive microelectrodes enabled us to follow the dynamic changes in extracellular pH (pHe) together with those in the extracellular concentration of some biologically important ions, particularly K+ and Ca2+. Activity-related changes in pHe were studied in isolated spinal cords of frogs and in spinal cords of rats in vivo. Repetitive electrical stimulation of an afferent input led either to triphasic alkaline-acid-acid changes (90% of frogs) or to triphasic alkaline-acid-alkaline changes (10% of frogs and rats) with the greatest changes in the lower dorsal horns. The transient acid shift by as much as 0.15-0.25 pH units is dominant and builds up during the stimulation. The changes in pHe were also found in response to various adequate stimuli applied to the skin on the hind limb. Using specific inhibitors of Na+/H+ exchange, K+-Cl- co-transport, Cl-/HCO3- exchange, the Na+/K+ pump and carbonic anhydrase, we found pHe homeostasis to be impaired and stimulation-induced changes in pHe decreased. We conclude that the pHe changes evoked by electrical or adequate stimulation of an afferent input are not determined by changes in extracellular strong ion concentration differences due to accumulation of lactate, since we found no effect of NaF, a metabolic blocker of lactate production. However, lactate accumulation has been demonstrated during seizures, spreading depression and anoxia. Recently, it has been recognized that the observed pHe changes can affect permeability of membrane ionic channels, neuronal excitability and glial cell function.

TiO 2 nanostructured films on mica using liquid phase deposition

Liquid phase deposition is a method to grow conformal TiO 2 films with good crystallinity on various substrates. The growth depends on the hydrophilicity of the substrate surface and we found that mica is a substrate capable of forming uniform and thick films. Films were grown using an aqueous solution of TiF 4 through a hydrolysis–condensation process. We have studied films properties; morphology, porosity and thickness, in terms of growth conditions, i.e. TiF 4 concentration, growth temperature, deposition time and pH. The porosity of films was shown to rapidly increase from about 18% to about 25% and remained the same for longer growth times. The growth process of films could be identified by three stages; the initial delay, the fast growth and finally growth saturation. The fast growth showed a first order kinetics and resulted in 0.086 eV activation energy that was attributed to Ti–F hydrolysis reaction. The growth saturation was at a mass thickness of about 500 nm and for low-temperature solutions could not be reached even after 70 h. We observed also a dynamic pH change during the growth, and attributed the saturation of growth to the drop of pH to values where growth speed is negligible.

Fentanyl Effervescent Buccal Tablets

Noninvasive opioid delivery systems that provide rapid pain relief offer advantages for outpatients with high-intensity, rapid-onset pain. fentanyl effervescent buccal tablets (FEBT) employ a novel delivery system that uses an effervescence reaction to enhance the rate and efficiency of oral transmucosal fentanyl absorption. Enhanced absorption of fentanyl is considered to be caused primarily by dynamic shifts in pH within the microenvironment lying between a dissolving tablet and the buccal mucosa. The initial drop in pH is a result of the dissolution of citric acid, which combines with bicarbonate to form carbonic acid. Carbonic acid dissociates into carbon dioxide and water. As carbon dioxide dissipates, the pH increases. The initial drop in pH favors the dissolution of fentanyl citrate, the ionized form of fentanyl, whereas the subsequent increase in pH favors the formation of nonionized fentanyl. Nonionized fentanyl prefers to associate with the more lipophilic molecules in the buccal mucosa rather than stay in solution surrounded by water molecules. The dynamic changes in pH that occur on the surface of FEBT as they dissolve can be measured in vitro. Pharmacokinetic studies in healthy volunteers have demonstrated more rapid and more complete fentanyl absorption with effervescent buccal delivery compared with noneffervescent buccal delivery. Controlled clinical studies in patients with high-intensity, rapid-onset pain are the next step in defining the clinical utility of this enhanced buccal absorption of fentanyl.

Ce4+-stimulated ion fluxes are responsible for apoptosis and taxol biosynthesis in suspension cultures of Taxus cells

Ion fluxes across the plasma membrane activated by 1 mM Ce4+, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce4+, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca2+ concentration decreased within the first 3 h after the addition of Ce4+, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca2+-channel blocker indicated that the dynamic changes in extracellular pH and the Ca2+ concentration resulted from the Ce4+-induced activation of H+ uptake and Ca2+ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce4+-treated cells to undergo necrosis, and the prior addition of the Ca2+-channel blocker inhibited Ce4+-induced taxol biosynthesis and apoptosis. It is thus inferred that H+ uptake is necessary for cells to survive a Ce4+-caused acidic environment and is one of the mechanisms of Ce4-induced apoptosis. Furthermore, the Ca2+ influx across the plasma membrane mediated both the Ce4+-induced apoptosis and taxol biosynthesis.