Bretylium causes a K(+)-Na+ pump activation that is independent of Na+/H+ exchange in depolarized rat, mouse and human lymphocytes.

We have studied a bretylium tosylate induced increase of the membrane potentials of partially depolarized rat, mouse and human lymphocytes, using the potential sensitive dye, bis [1,3, dibutylbarbituric acid-(5) trimethine oxonol]. The extent of this repolarization is dose-dependent and decreased in magnitude as the temp was reduced from 37 degrees C to room temp. The repolarizing effect is inhibited by K(+)-Na(+)-pump blockers or lack of extracellular Na+. Sodium ion channel blockers are effective in abolishing repolarization only if applied prior to, or simultaneously with, bretylium. Activation of Na+/H+ exchange is not involved in the mechanism of the phenomenon as the latter is completely eliminated in the presence of 10 microM amiloride (concn of the diuretics having no measurable inhibition on the action of the exchanger). These data suggest that bretylium opens ligand- and voltage-gated Na+ channels, and repolarization occurs due to higher activity of the K(+)-Na(+)-pump stimulated by the enhanced intracellular Na+ accumulation.

Hypo-osmotic shock induces an osmolality-dependent permeabilization and structural changes in the membrane of carp sperm.

We carried out spectrofluorimetric and flow cytometric measurements to investigate the effect of hypo-osmotic shock on cell membranes of common carp sperm. The time course of the permeability of the sperm cell membrane, as monitored by DNA-related propidium iodide (PI) fluorescence, was followed for 30 min after dilution of semen in hypo-osmotic environments of different ionic strengths. Spectrofluorimetric measurements indicated a continuous increase in the total PI emission intensity of a sperm suspension. Cell-by-cell flow cytometric measurements suggested that the permeability changes were of the all-or-none type. The permeabilized fraction of cells in the individual samples was time and osmolality dependent. The number and percentage of cells in which DNA was stained by PI increased gradually over time and reached a steady-state plateau value after 5-15 min. This equilibrium fraction of cells with a PI-permeable cytoplasmic membrane displayed an inverse relationship with the osmolality of the diluent, having a near 100% value for fresh water and distilled water. Dilution of sperm in hypo-osmotic medium brought about a fast decrease in the forward light-scattering signal on a short time scale compared to the pre-steady-state time of the permeabilization. With the addition of extracellular Ca2+ (1.8 mM), restoration of the light scattering signal was observed. Permeabilization of the membrane and restoration of light scattering were not coincident in time. We propose a two-dimensional reorganization of the lipid structure as the underlying mechanism of the latter process.

Cerebral effects of a single dose of intravenous vinpocetine in chronic stroke patients: a PET study.

The effects of vinpocetine (Cavinton) on the cerebral glucose metabolism of chronic stroke patients are studied with positron emission tomography. The regional and global cerebral metabolic rates of glucose (CMRglu) and the kinetic constants related to them are quantified before and after single-dose intravenous vinpocetine treatment. These measurements are completed with transcranial Doppler sonography and single photon emission computed tomography to explore the possible mechanisms underlying the resulting changes in glucose uptake and metabolism in the brain. The authors' findings indicate that a single-dose vinpocetine treatment, although it does not affect significantly the regional or global metabolic rates of glucose, improves significantly the transport of glucose (both uptake and release) through the blood-brain barrier in the whole brain, the entire contralateral hemisphere, and in the brain tissue around the infarct area of the symptomatic hemisphere. These changes are in accord with increased blood flow in the entire contralateral hemisphere as well as decreased blood flow velocity and increased peripheral vessel resistance in the entire symptomatic hemisphere.

A novel method for measuring intracellular pH: effect of neutron irradiation on pHi of transformed cells.

A new flow cytometric method was developed for measuring the intracellular pH (pHi) of mammalian cells using a fluorescent pH indicator dye 2',7-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). Emission intensities (or their ratios) measured from BCECF-loaded cells can be converted into absolute pHi values using appropriate calibration curves. By comparison of several possible measuring and data evaluation procedures a double-ratio method was suggested as the most advantageous protocol to yield reliable intracellular pH data. This method allows pHi to be determined on a cell-by-cell basis corrected for cell volume and change in geometry of input-output optics of the flow cytometer. Our method applies a standard calibration curve and does not necessitate its reconstruction for each new set of measurements. Cells of the OKT-4 and OKT-8 hybridoma lines were exposed to neutron irradiation of different doses. Irradiated cells underwent a biphasic alkalinization; an instantaneous effect detected within 1.5 h was found to be intensified over 24 h. For the interpretation of data we suggest that the increase in cytoplasmic pH following neutron treatment is evoked by two mechanisms.

Flow cytometric determination of absolute membrane potential of cells.

Membrane potential measurements using fluorescent membrane potential indicator dyes report on relative changes but usually do not result in an absolute value of the measured parameter. The method developed in this paper is based on the assumption that the negatively charged bis-oxonol distributes across the cytoplasmic membrane according to the Nernst equation. It is further supposed that the fluorescence intensity measured from a given stained cell is a single-value function of the intracellular dye concentration. The protocol suggested incorporates the construction of a calibration curve (fluorescence intensity measured from stained cells vs. extracellular dye concentration). This allows the evaluation of the membrane potential in millivolts using fluorescence readings of the cells both in the depolarized state and in the state of interest. Good agreement was found between absolute membrane potential data of human peripheral blood lymphocytes by our method and results of parallel patch clamp measurements.

Intracellular pH does not affect drug extrusion by P-glycoprotein.

The intracellular pH (pH(i)) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-V1/KB-3-1 system in this paper. The possible role of an elevated pH(i) in Pgp-related MDR has been investigated by shifting back the pH(i) of the MDR+ cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked delta pH(i) on relative daunorubicin (DNR) accumulation was similar in the case of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pH(i) on Pgp pumping efficiency. However, an indirect connection between pH(i) regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR+ cells.

Preparation and primary evaluation of [11C]CSC as a possible tracer for mapping adenosine A2A receptors by PET.

A 11C labeled selective adenosine A2A antagonist, (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[11C]methylxanthine [11C]CSC) was prepared by the reaction of (E)-8-(3-chlorostyryl)-1,3-dimethylxanthine and [11C]methyl iodide. The decay-corrected radiochemical yield was 32.3% with a radiochemical purity of 99%, a specific activity of 1.85-5.55 GBq/mumol and a preparation time of 1 h. A primary evaluation of [11C]CSC as a potential tracer for mapping adenosine A2A receptors by positron emission tomography (PET) is also presented. Biodistribution and autoradiographic studies were carried out on Swiss mice and domestic rabbits. In mice the lung showed the highest uptake at 10 min after i.v. injection, followed by the liver, kidney, heart and brain. Inside the brain a high level of radioactivity accumulated in the striatum, in accordance with previous findings on the specific spatial distribution of A2A adenosine receptors and also in the medulla oblongata. Dynamic PET studies on rabbits showed a fast brain uptake of CSC, reaching a maximum in less then 2 min. On the basis of competition experiments with the unlabeled ligand [11C]CSC proves to bind specifically to the appropriate receptor.

Regional glucose metabolic rates in the human brain: a PET study.

With the purpose of determining the regional cerebral glucose metabolic rates (rCMRglu) in the normal human brain in resting state, we measured with PET and FOG, as the tracer, the distribution of regional radioactivity levels in the brains of seven young healthy volunteers. The individual brain images were stereotactically standardised with the help of a computerised brain atlas. In the next steps, the global cerebral glucose metabolic rate (gCMRglu) in the individual subjects was normalised to 34 mumol/ 100 g/min, followed by the averaging of the stereotactically standardised and metabolically normalised brain images across the subject population. In the final step, on the basis of high precision anatomical information, three-dimensional (3-D) volumes-of-interest (VOI's), covering over thirty anatomical structures in the brain, were created and the rCMRglu values inside the VOI's were determined in the averaged image. The present measurements of the rCMRglu values are in outstanding agreement with those determined in earlier quantitative PET-FDG studies, demonstrating that stereotactically standardised and metabolically normalised images of regional radioactivity distributions in the brain can be used for quantitative determination of rCMRglu values.

Hypoglycemia activates compensatory mechanism of glucose metabolism of brain.

The effect of plasma glucose concentration on the cerebral uptake of [18F]-fluorodeoxy-D-glucose (FDG) was studied in a broad concentration range in a rabbit brain model using dynamic FDG PET measurements. Hypoglycemic and hyperglycemic conditions were maintained by manipulating plasma glucose applying i.v. glucose or insulin load. FDG utilization (K) and cerebral glucose metabolic rate (CGMR) were evaluated in a plasma glucose concentration range between 0.5 mM and 26 mM from the kinetic constant k1, k2, k3 obtained by the Sokoloff model of FDG accumulation. A decreasing set of standard FDG uptake values found with increasing blood glucose concentration was explained by competition between the plasma glucose and the radiopharmacon FDG. A similar trend was observed for the forward kinetic constants k1, and k3 in the entire concentration range studied. The same decreasing tendency of k2 was of a smaller magnitude and was reverted at the lowest glucose concentrations where a pronounced decrease of this backward transport rate constant was detected. Our kinetic data indicate a modulation of the kinetics of carbohydrate metabolism by the blood glucose concentration and report on a special mechanism compensating for the low glucose supply under conditions of extremely low blood glucose level.

[Comparative analysis of quantifying methods in FDG-PET scans of the brain]. / Agyi FDG-PET-vizsgálatok mennyiségi értékeló módszereinek összehasonlítása.

The comparative analysis of three tracer kinetic methods most frequently applied for the quantization of the results of FDG-PET-brain scans was performed. The data of measurements on five healthy patients were evaluated by the most general method developed by Phelps, the Patlak-procedure and the SUV- (standard uptake value) method. It was demonstrated by the aid of correlation analysis that the applicability of the results of the more simple methods to estimate glucose metabolic rate (GMR) as calculated by the Phelps-method depended on the kind of the selected region of the brain. It was shown that the most considerable distortion occurred in the case of the same anatomical regions of the brain with both simplified methods. These regions were located either in the white matter or in the vicinity of larger size blood vessels or they were elements of the base of the skull [gyrus rectus (l. u.), pons, capsula interna (l. u.), cerebellum (l. u.), corpus callosum]. The distorted estimation is explained by the fact that the simpler models neglect dephosphorylation of the FDG-6P, and they also disregard the contribution of the intravascular activity to the tissue radioactivity as determined by the relatively low resolution PET measurement. The correlation coefficient between the GMR as calculated by the Phelps-method and glucose consumption data by the investigated simpler methods had very low values for regions located in the white matter, eventual close to blood vessels or being elements of the base of the skull.

[11C-methionine: an effective radio-tracer for PET scan in the detection of tumors of low proliferating capacity]. / 11C-metionin: hatékony radiofarmakon az alacsony proliferatív kapacitású daganatok PET-vizsgálatához.

11C-methyl-methionine is available at the PET Center of University Medical School Debrecen since June, 1996. The first 5 oncological examinations were indicated for clinically suspected recurrent/residual tumorous tissue of low-grade/low proliferative capacity, following the negative or inconclusive results of previous 18F-deoxyglucose (FDG) examinations. In these situations, the methionine examinations provided conclusive results in 4 cases (out of the total of 5 examinations). On the basis of published data and own experience, the authors recommend methionine PET investigations for diagnosis, differential diagnosis and therapy monitoring of tumours of low-grade/low proliferative capacity following inconclusive results of previous FDG examination.

[Evaluation of the nature of brain tumors using methods of nuclear medicine]. / Agydaganatok természetének vizsgálata nukleáris medicinai módszerekkel.

FDG-PET studies permit an assessment of the degree of brain tumour malignancy and detection of tumour recurrence. MIBI-SPECT also affords promising results in this respect. In this work, the diagnostic value of MIBI-SPECT was compared with that of FDG-PET for the determination of primary brain tumours malignancy and the detection of recurrent brain tumours. SPECT and PET examination were carried out within a week in 14 patients (12 males, 2 females, mean age: 40 years, range 16-61 years) with brain tumours. Seven patients had a primary tumour, and in a further 7 MRI or the clinical signs and symptoms let to a suspicion of tumour recurrence. All tumours were verified histologically to be gliomas of grades I-IV. The SPECT and PET images were analysed visually and semiquantitatively. In 3 of the investigated 7 primary glioma patients, there was a visibly enhanced MIBI-positive cases, only one had an increased FDG uptake. In 4 of the 7 tumour recurrence cases, either the MIBI or the FDG uptake was visibly increased. All of these were histologically high-grade gliomas. In the remaining low grade tumours (primary of recurrent), neither MIBI nor FDG revealed a pathologically increased uptake. The intensity of radiopharmaceutical uptake at the site of the tumours was visually and semiquantitatively higher for MIBI that for FDG. It is concluded that MIBI-SPECT is a valuable and simple tool for evaluation of the biological characteristics of brain tumours, showing increased uptake of MIBI according to the malignancy and tumour recurrence of brain tumours.

[The effect of a single-dose intravenous vinpocetine on brain metabolism in patients with ischemic stroke]. / Egyszeri vinpocetin-infúzió agyi anyagcserére gyakorolt hatásának vizsgálata territoriális típusú ischaemiás stroke-ot szenvedett betegeken.

The effect of a single-dose i.v. infusion of vinpocetine on the cerebral blood flow (CBF) and glucose metabolism of post-stroke patients was studied by measuring the regional and global cerebral metabolic rates of glucose (CMRglu) and the corresponding kinetic constants before and after treatment. Transcranial Doppler (TCD) and single photon emission tomography (SPECT) measurements were also performed. The cerebral glucose metabolism was significantly higher in the contralateral hemisphere than in the affected one before therapy. In the affected hemisphere the regional glucose metabolism was inhomogenous: relatively low values were measured in the stroke region, whereas it was increased in the peristroke region. Although a single-dose vinpocetine treatment did not affect significantly the regional or global metabolic rates of glucose, the glucose transport (both intracellular up-take and release) was strongly affected in the whole brain, in the contralateral hemisphere and in the peri-infarct area of the symptomatic hemisphere. A slightly increased (not significant, N. S.) cerebral blood flow could be observed in the contralateral and a decreased flow (N. S.) in the symptomatic hemisphere.

Membrane permeability changes induce hyperpolarization in transformed lymphoid cells under high-density culture conditions.

BACKGROUND: Membrane potential changes in cells from the human lymphoid B cell line, JY, evoked by increasing cell density in culture were investigated, as data published on other cell types are controversial. An attempt was also made to clear the underlying mechanism. METHODS: Nonadherent JY cells were isolated from high-density plateau-phase cultures (type A cells), medium-density log-phase cultures (type B cells), and low-density lag-phase cultures (type C cells). They were analyzed for transmembrane potential, intracellular free concentration of potassium and sodium, membrane permeability for monovalent cations, cell cycle distribution by measuring DNA content, and glucose uptake. RESULTS: C type cells proved to be relatively depolarized (-41 +/- 3 mV) and cells obtained from the highest density cultures hyperpolarized (-60 +/- 3 mV). Intracellular concentrations ([K](i) = 92-97 mM and [Na](i) = 34-35 mM) were almost identical for each type of cell. The sodium/potassium permeability constant ratio in the A and C type of cells was 0.047 and 0.094, respectively. High-density culture conditions resulted in a pronounced G(1)-phase arrest. CONCLUSIONS: Differences in the membrane potential values induced by high-density culture conditions were maintained by changes in the membrane permeability for the monovalent cations.

Ligand and voltage gated sodium channels may regulate electrogenic pump activity in human, mouse and rat lymphocytes.

Bretylium tosylate - a sodium channel opener - resulted in an increase of membrane potential of depolarized human, rat and mouse T and B lymphocytes. Flow cytometric membrane potential measurements with bis-oxonol revealed that the above hyperpolarizing effect was amiloride, ouabain, tetrodotoxin, azide and temperature sensitive. The effect showed an absolute dependence on the extracellular sodium but it was insensitive to the extracellular Ca2+ level. The voltage gating of the effect can be eliminated by either an increase of the extracellular potassium concentration or low doses of veratrin. The existence of a voltage and ligand gated sodium channel is suggested in the plasma membrane of all kinds of lymphocytes. The hyperpolarization is explained by an increased activity of the electrogenic sodium-potassium ATP-ase. Induced opening of such sodium channels may regulate the electrogenic pump activity and indirectly cell activation.

Role of extracellular and intracellular pH in carp sperm motility and modifications by hyperosmosis of regulation of the Na+/H+ exchanger.

Recently developed flow cytometric methods (Trón et al.: Mol Immunol 27:1307-1311, 1990) to measure the intracellular pH (pHi) and intracellular potassium concentration in mammalian cells by using the fluorescent pH-indicator dye 2',7bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF) were adopted for measuring these parameters in carp sperm. The intracellular potassium concentration of the carp sperm was 62.4 +/- 5.3 mM. This is very similar to the potassium concentration of the seminal plasma (87 +/- 16 mM), and it suggests a depolarized state of the sperm cell in the semen. An average pHi value of 7.06 +/- 0.11 was obtained by measuring sperm samples taken from ten animals. Changes in the ionic composition of the environment did not alter pHi. Sperm motility was initiated by transferring the cells to an environment of 110 mOsm osmolality. This hypoosmotic shock induced fast changes in the membrane structure that could be reversed by restoring physiologic osmolality. Activation was accompanied by a fast alkalinization of the sperm cells. This pH change was amiloride sensitive, suggesting the involvement of the Na+/H+ exchanger in the activation process. Alkalinization of acid-loaded sperm cells depended on the osmolality of the environment. Equilibrium pHi of these cells in hyperosmotic buffers was substantially lower relative to cells in an isoosmotic environment. Effects of the extracellular and intracellular pH on carp sperm motility were also examined. Extracellular pH below 5.5 abolished sperm motility completely. Alkaline extracellular pH did not alter the duration of sperm motility even at extreme values (pHe = 9.6). Duration of the flagellar motion did not depend on the pHi between values of 6.5 and 8.5; however, it was significantly reduced both below and above this range. No motility was observed below pHi = 6.0 or above pHi = 9.5 with a 10 min incubation time at these pH values prior to activation. Effects of the extracellular and intracellular pH on sperm motility were partially reversible.

Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ influx and subsequent initiation of sperm motility in the common carp.

Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca(2+)-free hypoosmotic solutions, and significant increase in the intracellular Ca(2+) level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca(2+). Ca(2+) channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca(2+) influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca(2+) channels, leading to Ca(2+) influx at the initiation of carp sperm motility.

Flow cytometric determination of intracellular free potassium concentration.

A flow cytometric assay has been developed for determination of intracellular free potassium concentration ([K+]i). Investigated cells, loaded with the fluorescent pH indicator 2',7bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), are incubated in the presence of nigericin, and the intracellular pH is measured. The ionophore maintains the same ratio value [H+]/[K+] on both sides of the cytoplasmic membrane, so [K+]i can be evaluated from the measured intracellular pH (pHi) and the known parameters of the buffer. Application of the method revealed that the intracellular potassium concentration is significantly higher in lymphocytes than in proliferating cells of HUT-78, U266, and JY cell lines. A surprisingly low (60 mM) [K+]i concentration was observed with sperm cells of common carp. This method allows measurements on individual cells, provides data of excellent statistics, and still does not require large amounts of material. These features offer remarkable advantages over other techniques used for intracellular K+ measurements, such as steady-state fluorescence, atom absorption photometry, or energy-dispersive x-ray analysis.