Lajos Trón

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.

Role of ion channels and membrane potential in the initiation of carp sperm motility

The exposure of freshly spawned, immotile carp sperm to hypoosmotic media triggers the initiation of calcium-dependent flagellar motility. Intracellular calcium concentration has been thought to be the critical component in motility initiation, possibly acting through a novel signalling pathway. The sensitivity of sperm cells to changes of osmolality of the environment raises the question whether a mechanoregulated osmosensitive calcium pathway is involved in the activation mechanism of carp sperm motility. The sperm cells are in a depolarized state in the seminal plasma (W = –2.6 ± 3 mV) and they hyperpolarize upon hypoosmosis-induced activation of motility (W = –29 ± 4 mV). The intracellular sodium [Na + ]i, potassium [K + ]i and calcium [Ca 2+ ]i ion concentrations were determined in quiescent cells, and at 20, 60 and 300 s after activation. The [Na + ]i and [K + ]i of the quiescent cells were similar to the [Na + ]e and [K + ]e of the seminal plasma. Following hypoosmotic shock-induced motility, both [Na + ]i and [K + ]i decreased to one-fourth of the initial concentration. The [Ca 2+ ]i doubled at initiation of the motility of the sperm cells and remained unchanged for 5 min. Bepridil (50–250 µM), a blocker of the Na + /Ca 2+ exchanger, blocked carp sperm motility reversibly. Gadolinium, a blocker of stretch-activated channels (10–20 µM), inhibited sperm motility in a dose-dependent manner and its effect was reversible. Hypoosmotic shock fluidized the membrane and gadolinium treatment made it more rigid in both quiescent cells and hypotonic shock treated but immotile sperm cells. Based on these observations, it is suggested that, besides the well-known function of potassium and calcium channels, stretch-induced conformational changes of membrane proteins are also involved in the sperm activation mechanism of common carp.

The possible role of F-18 FDG positron emission tomography in the differential diagnosis of focal pancreatic lesions

Purpose To compare the diagnostic values of different methods for the differentiation of malignant from benign pancreatic lesions. Methods In 22 patients with focal pancreatic lesions, the carbohydrate antigen (CA) 19-9 level was measured; abdominal ultrasound (US), computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), and F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) were performed; and the value of these methods were analyzed for their use in cancer diagnosis. Results Malignant lesions were identified in six patients and verified by surgery or clinical follow-up. The CA 19-9 level was elevated in four of the five patients examined (sensitivity, 80%). In all six cases, US and CT revealed hypoechogenic and hypodense areas (sensitivity, 100%). In one patient, ERCP was unsuccessful but yielded true-positive results in three others (sensitivity, 60%). The sensitivity of FDG PET was 100%. Sixteen focal cases of pancreatic disease proved to be benign. The CA 19-9 level was elevated in four of them (specificity, 73%). Hypoechogenic and hypodense areas were evident on US and CT in eight patients. The specificity of CT was 50% (8 of 16 cases). The specificity of US was 47% (7 of 15 cases). The specificity of successful ERCP was 92%. Fourteen negative FDG-PET results were truly negative. In two patients, however, the PET findings proved to be falsely positive (specificity, 88%). Conclusions FDG-PET is an effective tool to differentiate malignant from benign focal pancreatic lesions. In persons with focal pancreatic hypoechogenic or hypodense lesions detected by CT or US and an elevated CA 19-9 level, FDG PET should be the next step in the diagnostic strategy.

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.

A Review on Radiogenic Lhermitte's Sign *

Radiation myelopathy is a rare, but extremely serious side-effect of radiotherapy. Recovery from radiation-induced motor sequelae is rare, whereas, the regeneration of sensory losses is relatively frequent. Among the sensory radiogenic injuries of the spinal cord, Lhermitte’s sign (LS) is most frequent. This review describes the clinical picture and diagnostic imaging signs of radiogenic LS. There have been only a few studies on large patient groups with radiogenic LS, demonstrating a rate of occurrence of 3.6–13%, relating mainly to mantle irradiation or the radiotherapy of head and neck tumors. These cases typically manifest themselves 3 months following radiotherapy and gradually disappear within 6 months. Only 3 LS cases have been described in the English literature with extraordinarily severe symptoms lasting for more than 1 year. MRI, a sensitive tool in the detection of demyelination, failed to reveal any pathological sign accompanying radiogenic LS. However, positron emission tomography demonstrated increased [18F]fluorodeoxyglucose accumulation and [15O]butanol perfusion, but a negligible [11C]methionine uptake in the irradiated spinal cord segments in patients with long-standing LS. These imaging data are suggestive of a close direct relationship between the regional perfusion and metabolism of the spinal cord, very much like the situation in the brain. We postulate that an altered, energy-demanding conduction along the demyelinated axons of patients with chronic radiogenic LS may explain the increased metabolism and perfusion.

Radiation myelopathy with partial functional recovery: PET evidence of long-term increased metabolic activity of the spinal cord

Postoperative telecobalt irradiation was performed with a biologically effective extrapolated response dose of 165 Gy2 delivered to the spinal cord of a papillary thyroid cancer patient. Incomplete cervical transection developed, followed by a gradual functional improvement, which is still continuing 8 years after radiotherapy. Between the 6th and 8th years of the clinical course, positron emission tomography investigations demonstrated an increased 18F-deoxyglucose accumulation and (15)O-butanol perfusion, but negligible 11C-methionine uptake in the irradiated spinal cord segment. We suggest that the increased metabolism and perfusion, and the lack of detectable protein synthesis may be related to the increased energy demands of action potential conduction, due to the higher than normal density of sodium channels along demyelinated axons displaying restored conduction.

Angiography effectively supports the diagnosis of hepatic metastases in medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) belongs in the group of neuroendocrine tumors with early lymphatic and hepatic dissemination. A high rate of undetectable metastases is hypothesized to be responsible for the frequent mismatch between the apparent relatively small tumor burden and the elevated plasma tumor marker level.

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.