László Balkay

Potassium channels regulate hypo-osmotic shock-induced motility of common carp (Cyprinus carpio) sperm

Abstract The osmolality and composition of fish seminal plasma usually suppress sperm motility in the testis and sperm duct. Change in the osmolality of the environment at spawning activates flagellar motion of the sperm tail. The effect of inhibitors of anion and cation channels on active motion was investigated by a microscopic motility test. While sodium channel inhibitors (amiloride and tetrodotoxin) as well as anion channel inhibitors (DIDS and ethacrynic acid) did not affect motility, potassium channel inhibitors (4-AP, quinine, veratrine, verapamil) decreased the duration of flagellar motion or abolished motility completely. Potassium channel inhibitors exerted an effect on sperm motility depending on the dose and length of incubation. The voltage-activated K+ blocker 4-aminopiridine proved to be the most potent inhibitor. Its effect was completely reversible, indicating that the functional state of potassium channels is essential to the activation mechanism of sperm. Intracellular pH of the spermatozoa was measured by a fluorescent flow cytometric technique. Hypo-osmotic shock induced intracellular alkalinization of carp sperm. Increase in pHi was 0.15 pH units. In the presence of 4-AP the measured amplitude and kinetics of the pHi change was the same as in the absence of this inhibitor, arguing against a regulatory role of intracellular pH change.

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.

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.

Effects of vinpocetine on the redistribution of cerebral blood flow and glucose metabolism in chronic ischemic stroke patients: a PET study ☆

The pharmacological effects of the neuroprotective drug vinpocetine, administered intravenously in a 14-day long treatment regime, on the cerebral blood flow and cerebral glucose metabolism in chronic ischemic stroke patients (n=13) were studied with positron emission tomography in a double-blind design. The regional and global cerebral metabolic rates of glucose (CMRglc) and cerebral blood flow (CBF) as well as vital physiological parameters, clinical performance scales, and transcranial Doppler parameters were measured before and after the treatment period in patient groups treated with daily intravenous infusion with or without vinpocetine. While the global CMRglc values did not change markedly as a result of the infusion treatment with (n=6) or without (n=7) vinpocetine, the global CBF increased and regional CMRglc and CBF values showed marked changes in several brain structures in both cases, with more accentuated changes when the infusion contained vinpocetine. In the latter case the highest rCBF changes were observed in those structures in which the highest regional uptake of labelled vinpocetine was measured in other PET studies (thalamus and caudate nucleus: increases amounting to 36% and 37%, respectively). The findings indicate that a 2-week long intravenous vinpocetine treatment can contribute effectively to the redistribution of rCBF in chronic ischemic stroke patients. The effects are most pronounced in those brain regions with the highest uptake of the drug.

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.

Development of an FPGA-based data acquisition module for small animal PET

We report on the design of a data acquisition (DAQ) module for a small animal PET camera developed at our institutes. During the design an important guideline was to develop a system which is built up from strictly identical DAQ modules, and which has no built-in hardware limitation on the maximum number of modules. The developed DAQ module comprises of an LSO scintillator crystal block, a position sensitive PMT, analog signal conditioning circuits, a digitizer, an field programmable gate array (FPGA) for digital signal processing, and a communication module through which the collected data are sent to a cluster of computers for postprocessing and storage. Instead of implementing hardware coincidence detection between the modules, we attach a precise time stamp to each event in our design, and the coincidence is determined by the data collecting computers during postprocessing. The digital CFD algorithm implemented in the FPGA gives a time resolution of 2 ns FWHM for real detector signals

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.

Development of an FPGA-Based Data Acquisition Module for Small Animal PET

We report on the design of a DAQ module for a small animal PET camera developed at our institutes. During the design an important guideline was to develop a system which is built up from strictly identical DAQ modules, and which has no built-in hardware limitation on the maximum number of modules. The developed DAQ module comprises of an LSO scintillator crystal block, a position sensitive PMT, analog signal conditioning circuits, a digitizer, an FPGA for digital signal processing and a communication module through which the collected data is sent to a cluster of computers for post processing and storage. Instead of implementing hardware coincidence detection between the modules we attach a precise time-stamp to each event in our design, and the coincidence is determined by the data collecting computers during the post processing. The digital CFD algorithm implemented in the FPGA gives a time resolution of 2 to 3 ns FWHM for real detector signals

Ethernet based distributed data acquisition system for a small animal PET

We report on the design of a small animal PET scanner being developed at our institutes. The existing setup is the first version of the miniPET machine consisting of four detector modules. Each detector module consists of an 8times8 LSO scintillator crystal block, a position sensitive photomultiplier, a digitizer including a digital signal processing board and an Ethernet interface board. There is no hardware coincidence detection implemented in the system and coincidence is determined based on a time stamp attached to every event by a digital CFD algorithm. The algorithm is implemented in the digital signal processing board and generates a time stamp with a coincidence resolution of less than 2 ns. The data acquisition system is based on Ethernet network and is highly scalable in size and performance

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.