Effect of muscimol on the picrotoxinand bicuculline-induced delay in the desensitization of the GABA receptor/Cl− channel complex

Effects of picrotoxin and bicuculline on the muscimol-dependent³⁶Cl⁻ entry into synaptoneurosomes of the rat cerebral cortex are examined as well as desensitization of³⁶Cl⁻ entry at muscimol concentrations of 5 and 50 μM. At the 5 μM concentration (which is close to the muscimol IC₅₀), picrotoxin and bicuculline inhibited Cl⁻ entry into synaptoneurosomes and decreased the desensitization. At the 50 μM concentration, muscimol completely abolishes the bicuculline effects both on Cl⁻ entry and desensitization. Inhibition of Cl⁻ entry by picrotoxin is also abolished by 50 μM muscimol, whereas the picrotoxin-induced decrease in the desensitization rate is not. It is shown that both bicuculline effects result from inhibition of the GABA receptor, but the action of picrotoxin on the desensitization of Cl⁻ entry into synaptoneurosomes is not closely related to the functional activity of the GABA receptor/Cl⁻ channel complex. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8, pp. 144–147, August, 1996     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

In vitro measurements of optical properties of porcine brain using a novel compact device

Knowledge of the optical properties of tissues can be applied in numerous medical and scientific fields, including cancer diagnostics and therapy. There are many different ways of determining the optical properties of turbid media. The paper describes measurements of the optical properties of porcine brain tissue using novel instrumentation for simultaneous absorption and scattering characterisation of small turbid samples. Integrating sphere measurements are widely used as a reference method for determination of the optical properties of relatively thin turbid samples. However, this technique is associated with bulky equipment, complicated measuring techniques, interference compensation techniques and inconvenient sample handling. It is believed that the sphere for some applications can be replaced by a new, compact device, called the combined angular and spatially resolved head sensor, to measure the optical properties of thin turbid samples. The results compare very well with data obtained with an integrating sphere for well-defined samples. The instrument was shown to be accurate to within 12% for μ_a and 1% for μ _s ^′ in measurements of intralipid-ink samples. The corresponding variations of data were 17% and 2%, respectively. The reduced scattering coefficient for porcine white matter was measured to be 100 cm⁻¹ at 633 nm, and the value for coagulated brain tissue was 65 cm⁻¹. The corresponding absorption coefficients were 2 and 3 cm⁻¹, respectively.     

Removal of Out-of-Plane Fluorescence for Single Cell Visualization and Quantification in Cryo-Imaging

We developed a cryo-imaging system, which alternates between sectioning (10–40 μm) and imaging bright field and fluorescence block-face image volumes with micron-scale-resolution. For applications requiring single-cell detection of fluorescently labeled cells anywhere in a mouse, we are developing software for reduction of out-of-plane fluorescence. In mouse experiments, we imaged GFP-labeled cancer and stem cells, and cell-sized fluorescent microspheres. To remove out-of-plane fluorescence, we used a simplified model of light-tissue interaction whereby the next-image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing an objective function on subtracted images. Tissue-specific attenuation parameters [μ _T: heart (267 ± 47.6 cm⁻¹), liver (218 ± 27.1 cm⁻¹), brain (161 ± 27.4 cm⁻¹)] were found to be within the range of estimates in the literature. “Next-image” processing removed out-of-plane fluorescence equally well across multiple tissues (brain, kidney, liver, etc.), and analysis of 200 microsphere images gave 97 ± 2% reduction of out-of-plane fluorescence. Next-image processing greatly improved axial-resolution, enabled high quality 3D volume renderings, and improved automated enumeration of single cells by up to 24%. The method has been used to identify metastatic cancer sites, determine homing of stem cells to injury sites, and show microsphere distribution correlated with blood flow patterns.     

Electrolyte transport across the rabbit caecum in vitro

Electrolyte transport across rabbit caecal epithelium was investigated in vitro using conventional shortcircuiting and radioisotope techniques. In standard saline the caecum exhibited a relatively high short-circuit current (I _sc=4.4 μEq · cm⁻² · h⁻¹) and conductance (6.43 mS · cm⁻²). Both sodium and chloride were absorbed (J _net ^Na =6.40 andJ _net ^Cl =3.40 μEq · cm⁻² · h⁻¹) and potassium was secreted (J _net ^K =−0.5 μEq · cm⁻² · h⁻¹). Removal of Na⁺ abolishedI _sc andJ _net ^Cl whereas removal of Cl⁻ reducedJ _net ^Na to 2.92 μEq · cm⁻² · h⁻¹ but did not alterI _sc. In HCO ₃ ⁻ free salines containing 10⁻⁴ M acetazolamideJ _net ^Cl was abolished andJ _net ^Na andI _sc were reduced to 2.3 and 2.5 μEq · cm⁻² · h⁻¹ respectively. A positive residual ion flux (∼ 1 μEq · cm⁻² · h⁻¹) was detected in standard and Cl⁻-free salines but not in Na⁺-free or HCO ₃ ⁻ buffers. Mucosal amiloride (10⁻³ M) decreased net Na⁺ and Cl⁻ absorption but did not decreaseI _sc. Mucosal DIDS (10⁻⁴ M) decreasedJ _net ^Cl while mucosal bumetanide (10⁻⁴ M) did not affect any of the measured parameters. Finally, addition of theophylline (8 mM) stimulated Cl⁻ secretion and increasedI _sc. It is concluded that net sodium absorption by caecal epithelia occurs by both electrogenic and electroneutral mechanisms whereas net chloride absorption occurs only by an electroneutral process. Coupling of the absorptive fluxes of Na⁺ and Cl⁻ may result from Na⁺/H⁺ and Cl⁻/HCO ₃ ⁻ antiport systems in this tissue. Finally, it is proposed that up to half of theI _sc is due to a Na⁺-dependent secretion of bicarbonate ion.     

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca²⁺-activated Cl⁻ current (I _Cl,Ca) from Xenopus oocytes was investigated in excised “giant” membrane patches with voltage and Ca²⁺ concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca²⁺ concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca²⁺-dependent. The lag phase decreases from about 10 ms after a small Ca²⁺ concentration jump (0.1 μM) to less than 1 ms after a saturating concentration jump (55 μM). The rate constant of the rising phase is half-maximal at about 5 μM. At saturating Ca²⁺ concentrations, the rate constant is 400 to 500 s⁻¹. The Ca²⁺ dependence of the stationary current can be described by the Hill equation with n=2.3 and K _0.5=0.5 μM. The amplitude of the stationary current decreases after the excision of the membrane patch with t _1/2≈5 min (run-down). The activation kinetics of the current elicited by a Ca²⁺ concentration jump is not affected by the run-down phenomenon. At low Ca²⁺ concentration (0.3 μM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca²⁺ levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca²⁺-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.     

Nitric oxide synthase in bronchial epithelium and nitric oxide metabolites in the lungs of rats with bronchial asthma after fenoterol inhalation

Nitric oxide synthase of the bronchial epithelium and concentrations of nitric oxide metabolites (NO₂ ⁻ and NO₃ ⁻) in bronchoalveolar lavage fluids were measured in rats with bronchial asthma after fenoterol inhalation. It was suggested that nitric oxide-ergic mechanisms can mediate the effects of inhaled β₂-adrenergic agonists. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 176–179, August, 2000     

Bactericidal properties of a defocused Nd-YAG-laser beam

Nd-YAG laser radiation of 1.06 or 1.32 μ wavelength killed a proportion ofStaphylococcus aureus andEscherichia coli cells at power densities of 40 to 80 W/cm² and effected a 100% kill of these cells at a power density of 1000 W/cm². In blood agar, the radiation of 1.06 μ produced bactericidal effects to a depth twice that of the radiation of 1.32 μ. Laser radiation of these wavelengths did not cause elimination of R plasmids and had no effect on the antibiotic resistance of bacteria. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 654–656, June, 1996     

Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

Age dependence of skin autofluorescence

The nontryptophane intrinsic fluorescence of finger pad skin was studied in men aged 20–70 years in order to evaluate the possibility of using this parameter as a biomarker of aging. Linear correlation coefficients (r) between the fluorescence intensity and age varied from 0.50 to 0.66 for various skin sites. Age dependence of the mean value of fluorescence (F) measured on 4 fingers can be approximated by a second-degree polynomial:F=2.82−0.083T+0.0014T ² (r ₁=0.64,r ₂=0.71), whereT is chronological age in years. The proposed measurement of skin autofluorescence is a simple, noninvasive, rapid test for evaluating the aging of the skin in subjects over 40 with a high age-related determination (r ²=0.5). Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 351–353, March, 1999     

Optimal power-to-mass ratios when predicting flat and hill-climbing time-trial cycling

The purpose of this article was to establish whether previously reported oxygen-to-mass ratios, used to predict flat and hill-climbing cycling performance, extend to similar power-to-mass ratios incorporating other, often quick and convenient measures of power output recorded in the laboratory [maximum aerobic power (W _MAP), power output at ventilatory threshold (W _VT) and average power output (W _AVG) maintained during a 1 h performance test]. A proportional allometric model was used to predict the optimal power-to-mass ratios associated with cycling speeds during flat and hill-climbing cycling. The optimal models predicting flat time-trial cycling speeds were found to be (W _MAP m ^−0.48)^0.54, (W _VT m ^−0.48)^0.46 and (W _AVG m ^−0.34)^0.58 that explained 69.3, 59.1 and 96.3% of the variance in cycling speeds, respectively. Cross-validation results suggest that, in conjunction with body mass, W _MAP can provide an accurate and independent prediction of time-trial cycling, explaining 94.6% of the variance in cycling speeds with the standard deviation about the regression line, s=0.686 km h⁻¹. Based on these models, there is evidence to support that previously reported -to-mass ratios associated with flat cycling speed extend to other laboratory-recorded measures of power output (i.e. Wm ^−0.32). However, the power-function exponents (0.54, 0.46 and 0.58) would appear to conflict with the assumption that the cyclists’ speeds should be proportional to the cube root (0.33) of power demand/expended, a finding that could be explained by other confounding variables such as bicycle geometry, tractional resistance and/or the presence of a tailwind. The models predicting 6 and 12% hill-climbing cycling speeds were found to be proportional to (W _MAP m ^−0.91)^0.66, revealing a mass exponent, 0.91, that also supports previous research.     

Mechanisms compensating for altered resistance to respiration during muscular exercise in man

Alterations in respiratory parameters following the substitution of a helium-oxygen (He−O₂) or sulfur hexafluoride-oxygen (SF₆−O₂) mixture for air were analyzed during the first 10 respiratory cycles in human volunteers exposed to either of these mixtures for 3 min at rest and during forced respiration. Both at rest and during moderate physical exercise neither the volume of pulrnonary ventilation nor the partial carbon dioxide pressure differed significantly in the subjects breathing air, He−O₂, or SF₆−O₂. When the He−O₂ mixture was substituted for air, the forces developed by the inspiratory muscles, the work of breathing, the activity of the parasternal intercostal muscles, and the central inspiratory activity were all reduced, whereas substitution of the SF₆−O₂ mixture for air led to significant increases in these four parameters. It is concluded that compensatory responses of the respiratory system to altered density of the gaseous medium develop on the basis of the afferent impulse traffic from mechanoreceptors of the lungs and respiratory muscles and also on account of segmental reflexes and intrinsic properties of the muscle fibers themselves. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 247–251, September, 1995 Presented by A. D. Ado, Member of the Russian Academy of Medical Sciences     

Is the balance between skeletal muscular metabolic capacity and oxygen supply capacity the same in endurance trained and untrained subjects?

We attempted to test whether the balance between muscular metabolic capacity and oxygen supply capacity in endurance-trained athletes (ET) differs from that in a control group of normal physically active subjects by using exercises with different muscle masses. We compared maximal exercise in nine ET subjects [Maximal oxygen uptake (VO₂max) 64 ml kg⁻¹ min⁻¹ ± SD 4] and eight controls (VO₂max 46 ± 4 ml kg⁻¹ min⁻¹) during one-legged knee extensions (1-KE), two-legged knee extensions (2-KE) and bicycling. Maximal values for power output (P), VO₂max, concentration of blood lactate ([La⁻]), ventilation (VE), heart rate (HR), and arterial oxygen saturation of haemoglobin (SpO₂) were registered. P was 43 (2), 89 (3) and 298 (7) W (mean ± SE); and VO₂max: 1,387 (80), 2,234 (113) and 4,115 (150) ml min⁻¹) for controls in 1-KE, 2-KE and bicycling, respectively. The ET subjects achieved 126, 121 and 126% of the P of controls (p < 0.05) and 127, 124, and 117% of their VO₂max (p < 0.05). HR and [La⁻] were similar for both groups during all modes of exercise, while VE in ET was 147 and 114% of controls during 1-KE and bicycling, respectively. For mass-specific VO₂max (VO₂max divided by the calculated active muscle mass) during the different exercises, ET achieved 148, 141, and 150% of the controls’ values, respectively (p < 0.05). During bicycling, both groups achieved 37% of their mass-specific VO₂ during 1-KE. Finally we conclude that ET subjects have the same utilization of the muscular metabolic capacity during whole body exercise as active control subjects.     

A near infrared spectroscopy study investigating oxygen utilisation in hydrocephalic rats

Determination of hydrocephalus and its severity is important for optimal management of the condition. We have used near infrared spectroscopy (NIRS) to assess changes in concentrations of oxygenated (O₂Hb), deoxygenated (HHb), total haemoglobin (tHb) and cytochrome c oxidase (Caa₃) in normal and hydrocephalic Texas (HTx) rats in response to a 5 min head down tilt and a sodium pentobarbitone (NaPB) challenge. The former was used to test vascular responses and the latter to test metabolic responses. The haemoglobin oxygenation index (HbD) was derived which provides information regarding oxygen utilisation ([HbD]=[O₂Hb]−[HHb]). With the tilt challenge, a significant (P=0.001) difference was observed in [HbD] between normal (n=24) and hydrocephalic (n=14) rats (–3.50 (−6.00 to 0.00) μM cm⁻¹ and 7.50 (0.75 to 14.25) μM cm⁻¹, respectively). In another experiment we tested the response of ten rats to NaPB administration and observed a significant difference (P=0.008) in [Caa₃] between normal (n=5) and hydrocephalic (n=5) rats (−6.60 (−7.55 to −5.50) μM cm⁻¹ and −2.20 (−5.60 to −1.05) μM cm⁻¹, respectively). Coronal sections of these ten rat brains were analysed and significant (P<0.05) relationships were found between some of the NIRS parameters and cortical thickness or lateral ventricle area measurements. Our studies demonstrate that a significant difference in cerebral oxygenation and haemodynamics can be observed between normal and hydrocephalic HTx rats using NIRS.     

Direct measurement of the optical properties of human breast skin

The optical properties of thin tissue samples are obtained using goniometric apparatus. Direct measurements of the scattering of 633 nm light by 20–120 μm thick samples ofin vitro human breast epidermis and dermis are numerically integrated to obtain values for the scattering and absorption coefficients μ_s and μ_a, respectively, and the anisotropy parameter g. The total attenuation coefficient μ_t is obtained from transmission measurements. The Beer-Lambert law is found to be sufficiently valid for ≌100 μm thick samples to be used as the basis of the numerical integration. However, normalisation errors involved with the integration mean that only approximate values of μ_a could be obtained. Values of ≌40 mm⁻¹ are obtained for μ_t and μ_s, with the value of μ_a estimated as 1 mm⁻¹. The optical properties of human breast epidermis and dermis are found to be similar at 633 nm, and the two could be considered as a single layer for modelling light propagation at this wavelength. Further, the intersubject variability seen in this study suggests that a model that uses a range of values for each optical property may be more useful in a clinical situation.     

Oxygen uptake kinetics during severe intensity running and cycling

The purpose of this study was to investigate the effect of exercise mode on the characteristics of the oxygen uptake ( V̇O₂) response to exercise within the severe intensity domain. Twelve participants each performed a treadmill running test and a cycle ergometer test to fatigue at intensities selected to elicit a mode-specific V̇O₂max and to cause fatigue in ~5 min. The tests were at 234 (30) m·min⁻¹ and 251 (59) W, and times to fatigue were 297 (15) s and 298 (14) s, respectively. The overall rapidity of the V̇O₂response was influenced by exercise mode [ V̇O₂max was achieved after 115 (20) s in running versus 207 (36) s in cycling; p<0.01]. V̇O₂ responses were fit to a three-phase exponential model. The time constant of the primary phase was faster in treadmill tests than in cycle ergometer tests [14 (6) s versus 25 (4) s; p<0.01], and the amplitude of the primary phase was greater in running than in cycling when it was expressed in absolute terms [2327 (393) ml·min⁻¹ versus 2036 (301) ml·min⁻¹; p=0.02] but not when it was expressed as a percentage of the total increase in V̇O₂ [86 (6)% versus 82 (6)%; p=0.09]. When quantified as the difference between the end-exercise V̇O₂ and the V̇O₂ at 2 min, the amplitude of the slow component was ~40% smaller in running [177 (92) ml·min⁻¹ versus 299 (153) ml min⁻¹; p=0.03]. It is concluded that exercise modality affects the characteristics of the V̇O₂ response at equivalent intensities in the severe domain.     

Human Kir2.1 channel carries a transient outward potassium current with inward rectification

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K⁺ current with inward rectification (I _to.ir) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around −70 mV). The current exhibited a current–voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V _0.5) of availability of the current was −49.4 ± 2.1 mV at 5 mM K⁺ in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I _to.ir observed previously in native cardiac myocytes. Interestingly, inactivation of the I _to.ir was strongly slowed by increasing intracellular free Mg²⁺ (Mg²⁺ _i , from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg²⁺ _i . Inclusion of spermine (100 μM) in the pipette solution remarkably inhibited both the I _to.ir and steady-state current. These results demonstrate that the Mg²⁺ _i -dependent current carried by Kir2.1 likely is the molecular identity of I _to.ir observed previously in cardiac myocytes.     

Estimation of transmembrane potential of thymic lymphocytes during dexamethasone-induced apoptosis

The use of potential-sensitive fluorescent probes allowed us to estimate transmembrane potentials of the plasma (Δφ_p) and mitochondrial (Δφ_m) membranes of rat thymocytes, which were −58±3 mV and −169±7 mV, respectively. Dexamethasone-induced apoptosis led to a significant decrease in Δφ_p (by 55%) and Δφ_m (by 17%). This effects of dexamethasone was dose- and time-dependent. Changes in Δφ_m were greater and preceded those in Δφ_p. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 117–120, January, 1999     

Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intracellular pH in a mouse macula densa cell line (NE-MD)

Changes in the luminal NaCl concentration ([NaCl]) at the macula densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse macula densa cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. Intracellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na⁺–K⁺–2Cl⁻ cotransporter, or in a low (23 mM) Cl⁻ solution. Furosemide- and low Cl⁻-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na⁺/H⁺ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na⁺ solution. In conclusion, low [Cl⁻], but not low [Na⁺] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.