Low-sodium Medium Research Articles

Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury.

Hyponatraemia is defined as a serum sodium concentration of <135mEql/L and is the most common electrolyte disturbance in patients with chronic heart failure. We hypothesize that hyponatraemia may induce Ca2+ overload and enhance reactive oxygen species (ROS) production, which will exacerbate myocardial injury more than normonatraemia. We investigated the effect of hyponatraemia on the ability of the heart to recover from ischaemia/reperfusion episodes. Cardiomyocytes were obtained from 1- to 3-day-old Sprague Dawley rats. After isolation, cardiomyocytes were placed in Dulbecco's modified Eagle's medium (DMEM) containing low sodium concentration (110, 120, or 130mEq/L) or normal sodium concentration (140mEq/L) for 72hours. Exposure of cardiomyocytes to each of the low-sodium medium significantly increased both ROS and intracellular Ca2+ levels compared with the exposure to the normal-sodium medium. In vivo, 8-week-old male Sprague Dawley rats were divided into four groups: control group (Con), furosemide group (Fur), low-sodium diet group (Lsd) and both furosemide and low-sodium diet group (Fur+Lsd). The hearts subjected to global ischaemia exhibited considerable decrease in left ventricular developed pressure during reperfusion, and the size of infarcts induced by ischaemia/reperfusion significantly increased in the Fur, Lsd and Fur+Lsd compared with that in the Con. Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury by Ca2+ overload and increasing in ROS levels.

Cleaner production of ammonium poly-vanadate by membrane electrolysis of sodium vanadate solution: The effect of membrane materials and electrode arrangements

In this study, we put forward a clean and complete process of V2O5 production from vanadium-containing solutions. It features membrane electrolysis for V/Na separation and AMV precipitation. With the aid of membrane electrolysis, the vanadium and sodium in the leaching solution can be directly separated using membrane-electrolysis. Meanwhile, the NaOH in the cathode chamber can also be recycled for vanadium slag decomposition. The electrolysis systems and membrane types have been investigated. It was found that 3-chamber electrolysis was much more energy efficient than the traditional 2-chamber electrolysis, while F8080 membrane showed a better separation efficiency than N117, CMI7000, LF0014 membrane. The main electrolysis parameters were also investigated, and it appeared that high current density and electrolyte temperature, as well as low initial cathodic NaOH concentration were beneficial for the separation efficiency. Under the optimum electrolysis conditions the electricity consumption of producing 750 kg V2O5 and 1000 kg NaOH and was 2151 kW h, with a current efficiency of 92.9%. During the vanadium precipitation process, NH4VO3 was used as precipitant for the first time, avoiding the introduction of impurity ions, such as Cl−, SO42− or Ca2+, realizing zero waste generation by source. A low-sodium medium product of (NH4)2V6O16 was precipitated at pH between 2 and 4. The commercial quality V2O5 product could be obtained by calcination and the ammonium slat was recycled for precipitation. This method can achieve the clean production of V2O5 with no wastewater or solid waste generated through the whole process.

Видовые особенности негеномной реакции главных клеток собирательных трубок почек крыс и мышей на альдостерон

We have previously shown the fast nongenomic effect of aldosterone on intracellular sodium ([Na+]i) in the principal cells of rat kidney cortical collecting duct (CCD) in low sodium outer medium (14 mM NaCl). The method of fluorescence microscopy with the intracellular dye Sodium Green was used to study the rate of [Na+]i changes in response to external sodium shift (from 137 to 14 mM ) in Wistar rat and C57bl/6 mouse principal cells. In the presence of aldosterone (10 nM), the level of intracellular sodium in the rat principal cells was higher than in control at the low sodium medium (14 mM NaCl) (р˂0.05). In the principal cells of the mice kidney CCD, the response to a sharp change in the sodium level in the outer medium was significantly faster than we have found in rat, and the nongenomic response to aldosterone (10 nM) was weaker than in rats (р˂0.05). The data obtained for the first time demonstrated the differences in the fast nongenomic aldosterone effects in the principal cells of rat and mice CCD on intracellular sodium balance.

Effect of Low-Sodium, Choline-Based Semen Diluent on Viability of Bovine Spermatozoa Stored at 4&amp;amp;deg;C

This study evaluated sperm viability over time, after dilution and refrigerator storage of fresh semen extended in either synthetic cauda epididymal plasma (CEP2), or in a low sodium medium (CJ2) supplemented with either AlbuMAX or egg yolk. Semen collected weekly for 4 weeks from 4 bulls and assigned within bulls, across treatments. After extension in either CEP2, or CJ2 containing either egg yolk or AlbuMAX, semen was cooled to 4°C, and evaluated for 7 days. A computer assisted sperm analysis (CASA) system was used for sperm evaluation. Particular emphasis was placed on sperm motility since it is the single most important sperm parameter influencing bull fertility. Total and progressive motility of sperm in CEP2 and CJ2-AlbuMAX were similar (P = 0.85 and P = 0.23, respectively), but both were lower (P < 0.01) when compared to CJ2-yolk. Fewer sperm had rapid motility in CEP2 and CJ2-AlbuMAX compared to CJ2-yolk (P < 0.01). Sperm straightness and linearity were greater in CJ2-AlbuMAX and CJ2-yolk than in CEP2 (P < 0.01). Mean velocity (VAP) and linear velocity (VSL) were greater (P < 0.01) in CJ2-AlbuMAX than either CEP2 or CJ2-yolk. The calculated curvilinear velocity (VCL) of spermatozoa in CEP2 was lower than CJ2-AlbuMAX (P = 0.01), but similar with CJ2-yolk (P = 0.54). Overall, every sperm parameter measured by the CASA system was equal to or higher for sperm stored 7 days in CJ2 medium as compared with CEP2. The CJ2 extender supplemented with egg yolk is a viable alternative for storing fresh bovine semen.

Stimulus-evoked outer segment changes occur before the hyperpolarization of retinal photoreceptors.

Transient retinal phototropism (TRP) has been predominantly observed in rod photoreceptors activated by oblique visible light stimulation. Dynamic confocal microscopy and optical coherence tomography (OCT) have revealed rod outer segment (ROS) movement as the physical source of TRP. However, the physiological source of ROS movement is still not well understood. In this study, concurrent near-infrared imaging of TRP and electroretinogram (ERG) measurement of retinal electrophysiology revealed that ROS movement occurs before the onset of the ERG a-wave, which is known to reflect the hyperpolarization of retinal photoreceptors. Moreover, substitution of normal superfusing medium with low-sodium medium reversibly blocked the photoreceptor ERG a-wave, but largely preserved the stimulus-evoked ROS movements. Our experimental results and theoretical analysis indicate that early, disc-based stages of the phototransduction cascade, which occur before the hyperpolarization of retinal photoreceptors, contribute to the TRP associated ROS movement.

Hyponatraemia alters the biophysical properties of neuronal cells independently of osmolarity: a study on Ni(2+) -sensitive current involvement.

What is the central question of this study? Hyponatraemia, an electrolyte disorder encountered in hospitalized patients, can cause neurological symptoms usually attributed to a reduction in plasma osmolarity. Here, we investigated whether low [Na(+) ] per se can cause neuronal changes independent of osmolarity, focusing on involvement of the Na(+) -Ca(2+) exchanger. What is the main finding and its importance? We show that hyponatraemia per se causes alterations of neuronal properties. The novel finding of Na(+) -Ca(2+) exchanger involvement helps us to elucidate the volume regulation following hyponatraemia. This might have relevance in a translational perspective because Na(+) -Ca(2+) exchanger could be a target for novel therapies. Hyponatraemia is the most frequent electrolyte disorder encountered in hospitalized patients, and it can cause a wide variety of neurological symptoms. Most of the negative effects of this condition on neuronal cells are attributed to cell swelling because of the reduction of plasma osmolarity, although in hyponatraemia different membrane proteins are supposed to be involved in the conservation of neuronal volume. We have recently reported detrimental effects of hyponatraemia on two different neuronal cell lines, SK-N-AS and SH-SY5Y, independent of osmotic alterations. In this study we investigated, in the same cell lines, whether hyponatraemic conditions per se can cause electrophysiological alterations and whether these effects vary over time. Accordingly, we carried out experiments in low-sodium medium in either hyposmotic [Osm(-)] or isosmotic [Osm(+)] conditions, for a short (24h) or long time (7days). Using a patch pipette in voltage-clamp conditions, we recorded possible modifications of cell capacitance (Cm ) and membrane conductance (Gm ). Our results indicate that in both Osm(-) and Osm(+) medium, Cm and Gm show a similar increase, but such effects are dependent on the time in culture in different ways. Notably, regarding the possible mechanisms involved in the maintenance of Cm , Gm and Gm /Cm in Osm(+) conditions, we observed a greater contribution of the Na(+) -Ca(2+) exchanger compared with Osm(-) and control conditions. Overall, these novel electrophysiological results help us to understand the mechanisms of volume regulation after ionic perturbation. Our results might also have relevance in a translational perspective because the Na(+) -Ca(2+) exchanger can be considered a target for planning novel therapies.

Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30-90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na(+), while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na(+), K(+) and Cl(-) channels failed to prevent apoptosis or AVD.

Live birth of twins after IVF of oocytes that were cryopreserved almost 12 years before

A 45-year-old woman received embryos from IVF by intracytoplasmic sperm injection (ICSI) with her own oocytes that were cryopreserved (slow freezing in a low-sodium medium) 11years and 7 and a half months before, when she was 33years old. From seven metaphase-II oocytes thawed, five survived, four were fertilized after ICSI and two cleaving embryos were transferred on day 3. A diamniotic dichorionic term pregnancy was achieved, ending with the delivery of two healthy girls. As far as is known, this case represents, to date, the longest storage period of cryopreserved human oocytes resulting in a live birth.

2 year follow-up of children born after the transfer of embryos obtained by in vitro fertilization of human oocytes cryopreserved in low sodium medium

OBJECTIVE: To evaluate the growth and development of children born after the transfer of embryos obtained by in vitro fertilization of human oocytes cryopreserved in a choline-based freezing medium. DESIGN: Long term follow-up study in a medical institution. MATERIALS AND METHODS: Follow-up of 4 ICSI-singletons born between January 2000 and January 2004 in Halitus Medical Institute. Obstetric, peri- and post-natal data were collected and children were examined at 6, 12 and 24 months after birth. The four patients included in the study cryopreserved their oocytes (41 oocytes in total) between 1999 and 2003. Our oocyte cryopreservation program started in 1998. At the moment, 48 patients maintain 629 oocytes cryopreserved (13,1 oocytes/ patient); in 5 cases fertility preservation was the indication for oocyte cryopreservation. RESULTS: Four women, aged 30 ± 0.81(mean ± SD) gave birth to 4 singleton infants. Mean duration of female infertility was 5, 5 years (range 3 to 8 years). Patients had their 41 oocytes thawed; survival rate was: 41,4 % (17 oocytes), fertilization rate was 64,7% (11). Number of embryos transferred/ patient: 2, 4, 3 and 2 (2,75 ± 0,96). All transfers were performed on day 2. Mean birthweight of infants was 2870,75 ± 631 g (range: 2050 to 3550 g). There was 1 preterm delivery at 34 weeks of gestation; neonatal intensive care was required in this case. In all, 1 was a vaginal delivery and the other 3 were caesarean sections. Three healthy girls (75%) and one healthy baby boy (25%) were born. No perinatal mortality occurred. No post-partum complications were observed. At 6, 12 and 24 months of age, weights and heights of children were within normal parameters: mean weights: 7525, 10135 and 12562 g; and mean heights 63, 74,6 and 86,5 cm respectively. Neurological outcome of the four children at the age of 12 months showed normal development. CONCLUSIONS: The obstetric, peri- and post-natal outcome of the four babies was good. Further development of children was normal after 2 years of follow up. To our knowledge, this is the first report to present preliminary follow-up data of children born after oocyte cryopreservation in a low sodium medium. Careful evaluation is essential since choline based freezing technique is relatively new. These promising findings enhance the need for long-term follow-up of children born after this procedure.

256. Bovine oocyte vitrification in sodium free medium

Vitrification has been widely developed for the cryopreservation of mammalian embryos. Despite considerable effort, the vitrification of immature bovine oocytes is less successful with poor survival post thawing and low fertilization rates and development to offspring. The MII oocyte is highly sensitive to temperature changes with cryopreservation resulting in high incidence of aneuploidy after fertilization. Recently the concentration of sodium ions has been implicated in vitrification-induced damage. This study investigated the effect of a low-sodium choline-based medium (CJ2) supplemented with either 10% bovine fetal serum (FBS), 0.8% bovine serum albumin (BSA) or 1% polyvinylalcohol (PVA) on the ability of in vitro matured metaphase II (MII) oocytes to survive vitrification. Cumulus removed MII matured oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in CJ2 containing FBS, BSA or PVA for 5 minutes at 37ºC and then transferred into a vitrification solution composed of 20% EG, 20% DMSO and 0.6 M sucrose in CJ2. Twenty MII stage oocytes at a time were aspirated into the Gel Loader tip (GL-tip) with approximately 20 µL of vitrification solution, equilibrated 30 sec before plunging directly into liquid nitrogen and stored for 2 h. Oocytes were thawed rapidly and cryoprotectants removed by step-wise dilution in 0.25 M, 0.15 M, 0 M sucrose in TCM-199, 5 min each. Oocytes were then incubated in TCM-199 for 2 h before being stained with Hoechst-33342 and viewed under epi-florescence to determine survival. Developmental competence was determined by parthenogenetically activating (PA) surviving oocytes using calcium ionophore/6-dimethylaminopurine and cultured for 7 days in mSOF medium supplemented with 0.8% BSA. The recovery from vitrification procedure, survival post thawing and PA rates are summarized in the Table below. Vitrification in GL-tips allows efficient processing of MII oocytes with high rates of recovery. Overall, blastocyst development in this experiment was low in the control, sham groups and treated groups and that results may have been influenced by other factors (i.e. oocyte quality). Nevertheless, results demonstrate that reducing or eliminating sodium ions from the vitrification medium may protect the immature oocytes during vitrification or thawing and allow oocytes to be cryopreserved more effectively.

Induction of a Sodium-dependent Depolarization by External Calcium Removal in Human Sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.

Atypical relaxation by scorpion venom in the lamb urethral smooth muscle involves both NO-dependent and -independent responses.

The sustained depolarisation induced by alpha-toxins from scorpion venom (20 microg/ml(-1)) was used to test the hypothesis that an endogenous, photo-sensitive, nitrocompound could act as a stable nitrergic transmitter in the sheep (lamb) urethra. Scorpion venom-treatment effectively abolished neurogenic responses to electrical field stimulation, but it did not modify the spontaneous urethral photorelaxation. On the other hand, scorpion venom induced an atypical relaxation in noradrenaline-contracted preparations, which could be reverted, but not prevented, by tetrodotoxin (TTX, 1 microM). However, after TTX-pretreatment, relaxations elicited by scorpion venom were significantly delayed and slowed down, and similar responses were obtained in the presence of ouabain (10 microM), low sodium medium, or after the inhibition of the NO-cGMP pathway. Although the involvement of K(+) and Cl(-) channels can be ruled out since both charybdotoxin (300 nM) and chlorotoxin (50 nM) did not elicit any urethral relaxation nor modified the scorpion venom-induced one. However, a slow Ca(2+) channel seems to be involved. GVIA omega-conotoxin (1 microM), but not MVIIC omega-conotoxin (1 microM), significantly inhibited both EFS- and scorpion venom-induced relaxations and almost abolished the partial relaxation that was resistant to NO synthase inhibition. On the other hand, the presence of L-cis-diltiazem (0.3 mM), a selective inhibitor of cyclic nucleotide gated channels (CNGCs), also delayed and slowed down relaxation induced by scorpion venom, as well as abolish its reversal by TTX. L-cis-diltiazem pre-treatment induced a progressive decay in urethral relaxation brought about by electrical field stimulation only when repetitive, long duration stimulation protocols were used. Taken together, our results do not support the hypothesis of the endogenous, photo-sensitive, urethral nitrocompound as reflecting a stable nitrergic transmitter instead of NO. However, they suggest the involvement of both a NO-cGMP-dependent and TTX-sensitive component and a NO-independent response, mediated by GVIA omega-conotoxin-sensitive Ca(2+) channels, in the neurogenic relaxation of the urethral muscle. In addition, the likely involvement of CNGCs as an additional component of the cGMP signalling mechanism is suggested.

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium

Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.

Identification of a functional Na +/Mg 2+ exchanger in human trophoblast cells

Background Ionized magnesium levels are elevated in fetal blood compared with maternal blood, suggesting that the placenta may possess an active transport mechanism for magnesium. In the present study, we sought to determine the existence of an active transport mechanism for magnesium in the placenta using cultured trophoblast cells. Methods Using choriocarcinoma cells as a model system, we attempted to demonstrate the presence of a functional Na +/Mg 2+ exchanger. Human choriocarcinoma JEG-3 cells cultured on glass coverslips were loaded with MAG-Fura 2-AM (5 μmol/L × 30 min) to spectrofluorometrically assess kinetics of intracellular magnesium ([Mg 2+] i). Cells were superfused with various concentrations of Na +, Mg 2+, Ca 2+ and imipramine, a blocker of erythrocyte Na/Mg exchange. [Mg 2+] i calibration was determined via Triton X-100 and EDTA. Results: Sequential lowering of extracellular Na + caused progressively larger, transient increases in [Mg 2+] i. These transient changes in [Mg 2+] i were completely dependent on [Mg] o but was independent of extracellular calcium ([Ca] o). Although acute imipramine did not alter basal [Mg 2+] i, imipramine eliminated the return-to-basal phase of the [Mg 2+] i transient induced by low sodium medium. Increasing extracellular magnesium ([Mg] o) caused stepwise increases in [Mg 2+] i. Conclusions The JEG-3 cells appear to possess a functional Na/Mg exchanger that functions to maintain low [Mg 2+] i in cytotrophoblast cells. In addition, [Mg 2+] i is acutely regulated by [Mg] o. Because placental trophoblasts are sites of maternal–fetal ion exchange, and [Mg] o is altered in preeclampsia, derangements in or modulation of this exchanger may contribute to complications of pregnancy such as pregnancy-induced hypertension, preeclampsia, and preterm labor.

Angiotensin II induces inward currents in subfornical organ neurones of rats.

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.

Facilitation of plateau potentials in turtle motoneurones by a pathway dependent on calcium and calmodulin.

1. The involvement of intracellular calcium and calmodulin in the modulation of plateau potentials in motoneurones was investigated using intracellular recordings from a spinal cord slice preparation. 2. Chelation of intracellular calcium with BAPTA-AM or inactivation of calmodulin with W-7 or trifluoperazine reduced the amplitude of depolarization-induced plateau potentials. Inactivation of calmodulin also inhibited facilitation of plateau potentials by activation of group I metabotropic glutamate receptors or muscarinic receptors. 3. In low-sodium medium and in the presence of tetraethylammonium and tetrodotoxin, calcium action potentials evoked by depolarization were followed by a short hyperpolarization ascribed to the calcium-activated non-selective cationic current (ICAN) and by a dihydropyridine-sensitive afterdepolarization. The amplitude of the afterdepolarization depended on the number of calcium spikes and was mediated by L-type calcium channels. 4. The dihydropyridine-sensitive afterdepolarization induced by calcium spikes was reduced by blockade of calmodulin. 5. It is proposed that plateau potentials in spinal motoneurones are facilitated by activation of a calcium-calmodulin-dependent pathway.

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.

Endogenous Nucleosides in the Guinea-pig Eye: Analysis of Transport and Metabolites

This study investigates the transport of endogenous nucleosides and deoxynucleosides from the capillaries of the eye into the aqueous humour and the lens using thein situvascular eye perfusion technique in the guinea-pig. The transport of [3H] adenosine and [3H] thymidine across the blood-aqueous barrier proved to be very rapid with a volume of distribution after 4 minutes perfusion reaching 11.9±3.0% and 9.93±1.1%, respectively. However, the transport of [3H] guanosine and [3H] cytidine was slower, with volumes of distribution reaching only 3.38±0.58% and 4.8±1.41%. The values for the entry of deoxyadenosine and deoxyguanosine were not significantly different from the values obtained for corresponding ribonucleosides (adenosine and guanosine) so that a change in the pentose sugar does not change the affinity of the nucleoside for the transport protein. Perfusion with a low sodium medium inhibited the transport of [3H] adenosine and [3H] thymidine into the aqueous humour. The presence of 800 nMNBTI also caused a decrease in adenosine transport into the aqueous humour, so that the volume of distribution after 2 minutes reached only 3.78±1.87%. These findings suggest that the transfer of adenosine across the blood-aqueous barrier has both concentrative and equilibrative components. The presence of 0.1 mMthymidine had no effect on the [3H] adenosine transport, whereas 0.1 mMof adenosine resulted in a marked decrease on the [3H] thymidine transport which suggests that the concentrative nucleotide transport is probably mediated by bothcifandcittransport systems. The cellular uptake of nucleosides into the lens was very rapid and the volume of distribution of purine nucleosides was within the range of 30–50% whereas that for thymidine uptake was somewhat lower, reaching 20–30%. HPLC analysis of the eye structures in the guinea-pig showed that lens, vitreous body and the rest of the eye do not contain either free nucleosides or purine bases in detectable quantities, except for xanthine which was detected in aqueous humour at a concentration of 2.51±0.51 mM.However, serum of the anaesthetised guinea-pig did not contain xanthine in detectable amount so it seems that the metabolic degradation of the nucleosides in the guinea-pig eye progresses as far as xanthine, which is then accumulated in the aqueous humour.

Cloning of the cotA gene of Synechococcus PCC7942 and complementation of a cotA-less mutant of Synechocystis PCC6803 with chimeric genes of the two strains

cotA, a homologue of cemA that encodes a chloroplast envelope membrane protein, was cloned from Synechococcus PCC7942. The gene encodes a protein of 421 amino acids, which is similar in size to CotA of Synechocystis PCC6803 and CemA of liverwort and Chlamydomonas. There was significant sequence homology among these CotA and CemA in the C-terminal region but the homology was low in the N-terminal region. Sequencing of Synechococcus DNA in the cotA region revealed two other genes downstream of cotA, one of which is homologous to cobP and could be cotranscribed with cotA. A mutant (M48) was constructed by inactivating cotA in the wild-type (WT) Synechococcus. The mutant showed the same characteristics as the cotA-deletion mutant of Synechocystis (M29) and was unable to grow in a low sodium medium or at acidic pH under aeration with 3% CO2in air (v/v). Synechococcus cotA did not comple-ment M29. Three chimeric cotA genes of the two cyanobacterial strains were constructed. One of these chimeric genes strongly and the other two weakly complemented the mutant.