Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.     

Sodium current expression during postnatal development of rat outer hair cells

Outer hair cells of the cultured organ of Corti from newborn rats (0-11 days after birth) were studied in the whole-cell patch-clamp configuration. A voltage-activated sodium current was detected in 97% (n = 109) of the cells at 0-9 days after birth. The properties of this current were: (1) its activation and inactivation kinetics were fast and voltage-dependent, (2) the voltage at half-maximum activation was -45.0 mV, (3) its steady-state inactivation was temperature-sensitive (the half-inactivating voltage was -92.6 mV at 23 degrees C and -84.8 mV at 37 degrees C), (4) the reversal potential (80 mV) was close to the sodium equilibrium potential and currents could be abolished by the removal of extracellular sodium, and (5) tetrodotoxin blocked the current with a Kd of 474 nmol/l. Current amplitudes were up to 1.7 nA at room temperature. Mean current amplitudes showed a developmental time course with a maximum at postnatal days 3 and 7 for outer hair cells from the basal and apical part of the cochlea, respectively. In current-clamp mode cells had membrane potentials of -59.7 +/- 11.7 mV (n = 9). When cells were hyperpolarized by constant current injection, depolarizing currents were able to trigger action potentials. At 18 days after birth, sodium currents were greatly reduced and barely detectable. The results show that, unlike adult outer hair cells, immature outer hair cells regularly express voltage-gated sodium channels. However, due to mismatching of the sodium current inactivation range and membrane potential in vitro, a physiological function appears questionable.     

Sensitivity of blood pressure and renin activation during sodium restriction

The objective of the present study was to explore the interrelationships among cumulative sodium loss, renin activation, and blood pressure changes during sodium restriction in essential hypertensive patients. Specifically, we wanted to know whether the degree of sodium sensitivity of blood pressure depends on renin activation during steady state or on initial renin activation during the first days of sodium restriction. Sixty-seven untreated essential hypertensive patients were admitted to a metabolic ward for 8 days and put on a sodium restricted diet of 55 mmol/d from the second to the last day. Urinary excretions of sodium, potassium, and creatinine were determined along with mean arterial pressure and weight during 7 days. Besides measurements in steady state condition (after 7 days), active plasma renin concentration, aldosterone, and catecholamines were also assessed during the first 3 days of sodium restriction. Analyzable data are available for 55 patients. Baseline sodium excretion and the activation of renin during the first 3 days both appeared to be predictors of total sodium loss after 7 days. Changes in blood pressure were not related to changes in sodium balance, but they were to baseline blood pressure, baseline norepinephrine, and renin activation during the early phase of sodium restriction. In addition, blood pressure appeared to fall more when the normal relationship between sodium loss and early (but not late) activation of renin was disturbed. We conclude that sodium sensitivity of blood pressure during sodium restriction is associated with a relative unresponsiveness of the renin system during the early phase of sodium loss rather than to absolute renin levels during steady state.     

Hypernatriuria and kaliuresis in enuretic children and the diurnal variation

PURPOSE: We investigate the underlying pathophysiological cause of primary nocturnal enuresis by comparing electrolyte alterations in urine samples of enuretics during the daytime and nighttime compared with those of nonenuretic subjects. MATERIALS AND METHODS: Urine output, urine specific gravity and urinary electrolytes in 15 enuretic and 12 nonenuretic children were measured. We collected daytime serum and urine samples of children fed a similar diet between 7 a.m. and 7 p.m., and nighttime between samples 7 p.m. and 7 a.m. Urinary calcium/creatinine ratio, tubular reabsorption of phosphorus and excretions of fractional sodium and potassium were calculated. RESULTS: There was no significant difference between the calcium/creatinine ratio ratios. There was a significant increase in fractional sodium and fractional potassium values in enuretics compared to nonenuretics during the day and at night. Daytime and nighttime fractional sodium and fractional potassium values in enuretics were similar. In contrast to nonenuretics, enuretic patients had no diurnal variation of fractional sodium. There was significant positive correlation between bedwetting status, and fractional sodium and fractional potassium. CONCLUSIONS: Since sodium and potassium excretions were higher in enuretic patients than nonenuretic children, and no significant diurnal variation in urinary excretion of these ions there might be a difference in the mechanism of reabsorption of sodium and potassium between enuretic and nonenuretic children.     

Characterization of ionic currents of cells of the subfornical organ that project to the supraoptic nuclei

The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current.     

Lamotrigine may limit pathological excitation in the hippocampus by modulating a transient potassium outward current

Actions of the new antiepileptic drug lamotrigine were characterised using whole cell patch clamp recordings from rat CA1 pyramidal cells in vitro. The results suggest that lamotrigine, besides its previously described effect on the fast sodium inward current and calcium currents, modulates the transient potassium outward current ID. This may be an effective mechanism to inhibit pathological excitation.     

Developmental changes of potassium currents of embryonic leech ganglion cells in primary culture

The expression of calcium-activated potassium currents (IK(Ca)), delayed outward rectifier potassium currents (IK(slow)), and transient outward currents (IA) was studied during the development of the nervous system of the leech using the whole-cell patch-clamp recording technique. Dissociated cells were isolated from leech embryos between stage E7 and E16 and maintained in primary culture. K+ currents were recorded at E7, when only few anterior ganglia had formed beneath the primordial mouth. IK(slow) was present in all cells tested, while IK(Ca) was expressed in only 67% of the cells studied. Even as early as E7, different types of IK(Ca) have been found. Neither frequency of occurrence nor the charge density of IK(Ca) showed significant changes between E7 and E16. The density of IK(slow), however, increased by a factor of two between E7 and E8, which resulted in a significant increase in the total K+ current of these cells. This rise in potassium outward current developed in parallel with the appearance of Na+ and Ca2+ inward currents (Schirrmacher and Deitmer: J Exp Biol 155:435-453, 1991) during early development, shaping the electrical excitability in embryonic leech neurones. I(A) could be separated by its voltage-dependence and pharmacological properties. The current was detected at stage E9, when all 32 ganglia are formed in the embryo. The frequency of occurrence of I(A) increased from 16% at E9 to 70% at E15. The channel density, steady state inactivation, and kinetics showed no significant changes during development.     

Activation-inactivation coupling in Myxicola giant axons injected with tetraethylammonium

Myxicola giant axons internally injected with tetraethylammonium chloride to block potassium currents were examined under voltage clamp. The sodium inactivation time constants obtained from the decline in INa during step depolarizations were substantially smaller than those obtained using conditioning prepulses to the same potentials and the ratios agreed with previous observations using TTX. Inactivation shifts were also measured and found to be comparable to previous results.     

Zebrafish touch-insensitive mutants reveal an essential role for the developmental regulation of sodium current

Developmental changes in neuronal connectivity and membrane properties underlie the stage-specific appearance of embryonic behaviors. The behavioral response of embryonic zebrafish to tactile stimulation first appears at 27 hr postfertilization. Because the touch response requires the activation of mechanosensory Rohon-Beard neurons, we have used whole-cell recordings in semi-intact preparations to characterize Rohon-Beard cell electrical membrane properties in several touch-insensitive mutants and then to correlate the development of excitability in these cells with changes in wild-type behavior. Electrophysiological analysis of mechanosensory neurons of touch-insensitive zebrafish mutants indicates that in three mutant lines that have been examined the sodium current amplitudes are reduced, and action potentials either have diminished overshoots or are not generated. In macho mutants the action potential never overshoots, and the sodium current remains small; alligator and steifftier show similar but weaker effects. The effects are specific to sodium channel function; resting membrane potentials are unaffected, and outward currents of normal amplitude are present. Developmental analysis of sodium current expression in mechanosensory neurons of wild-type embryos indicates that, during the transition from a touch-insensitive to a touch-sensitive embryo, action potentials acquire larger overshoots and briefer durations as both sodium and potassium currents increase in amplitude. However, in macho touch-insensitive mutants, developmental changes in action potential overshoot and sodium current are absent despite the normal regulation of action potential duration and potassium current. Thus, the maturation of a voltage-dependent sodium current promotes a behavioral response to touch. A study of these mutants will allow insight into the genes controlling the maturation of the affected sodium current.     

微波消解-火焰原子吸收光谱法测定氮化铝粉中钾和钠

氮化铝粉体中钾、钠杂质含量对氮化铝陶瓷制品的热导率有重要影响。常温常压下氮化铝无法溶解,而碱熔法不适用于钾、钠的分析。实验以盐酸辅助微波消解分解氮化铝样品,并采用火焰原子吸收光谱法(FAAS)分别于波长766.5nm和589.0nm处测定钾、钠含量,建立了微波消解-火焰原子吸收光谱法测定氮化铝粉中钾和钠的方法。实验研究了铝基体及共存元素对钾、钠测定的影响,优化了微波消解条件、酸度以及消电离剂的加入量。钾、钠校准曲线线性相关系数分别为0.999 9和1.000,钾、钠特征浓度分别为0.026μg/mL和0.015μg/mL,钾、钠的检出限分别为0.7μg/g和0.5μg/g。按照实验方法测定氮化铝样品中钾、钠,结果的相对标准偏差(RSD,n=7)均小于4%;加标回收率分别为103%和98%;结果与电感耦合等离子体原子发射光谱法(ICP-AES)分析结果相吻合。方法以高纯铝进行基体匹配有效地解决了基体效应对测定的影响,适用于纯度大于99.9%的氮化铝粉中钾、钠的分析。     

Helping patients to die--an ethical challenge to nursing care and to its institutional framework]

The objective of this study was to determine fluid-electrolyte changes in male Wistar rats during 90 days of hypokinesia (decreased motor activity) and 15 days posthypokinesia. The animals were divided into two groups: rats subjected to hypokinesia served as experimental animals and rats placed under vivarium conditions served as control animals. The hypokinetic effect was carried out by keeping the experimental rats in small individual cages which restricted all their movements without hindering food and water intake. Determination was made of body weight, fluid consumed and eliminated in urine, sodium and potassium excretion in urine, concentrations of sodium and potassium in urine, the hematocrit level and water content in blood, and plasma concentration of sodium and potassium. During the experimental period body weight, water intake, urinary sodium and potassium content, and water content in blood decreased significantly, while electrolyte excretion in urine, plasma electrolyte concentration, hematocrit content and fluid excretion in urine increased significantly in the hypokinetic animals when compared with the control animals. During the initial seven days of the postexperimental period, water intake increased significantly while hematocrit level, water content in blood, and electrolyte plasma concentration remained markedly higher, and the fluid electrolyte excretion and electrolyte concentration thereof in urine decreased significantly. However, all these changes reverted back to the control level by the end of the post-experimental period. It was concluded that prolonged hypokinesia (HK) and the initial stages of post hypokinesia are associated with significant sodium and potassium changes and water consumption and elimination disturbances.     

Sodium and potassium metabolism in the dehydrated and rehydrated Bedouin camel

The effects of 10 days dehydration and rapid rehydration on the sodium and potassium metabolism in the one-humped camel were examined. The research was carried out during two periods in the summer, a cool and a hot period. In the hot period the effects of dehydration were found to be more severe. The potassium metabolism was more affected than that of the sodium. The concentrations of potassium in the urine declined while those of sodium increased. There were also marked changes in the filtered loads, excreted loads, and reabsorption of the two cations. Following rehydration significant changes in the cation metabolism were recorded within 15-45 min. It is suggested that antidiuretic hormone and not aldosterone caused the changes in the two-cation metabolism.     

Selective alteration of sodium channel gating by Australian funnel-web spider toxins

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.     

Sodium channels accumulate at the tips of injured axons

The axolemmal distribution of voltage-gated sodium channels largely determines the regions of axonal electrical excitability. Using a well-characterized anti-sodium channel antibody, we examined peripheral nerve fibers focally injured by exposure to the neurotoxic agent, potassium tellurite (K2TeO3). Immunocytochemical and radioimmunoassay data showed a focal accumulation of sodium channels within the tips of injured axons. The major increase in sodium channel concentration occurred between 7 and 11 days after toxin exposure; however, immunocytochemically, excess sodium channels persisted in several axonal endings for a much longer time. The accumulation of sodium channels at injured axonal tips may be responsible, in part, for ectopic axonal excitability and the resulting abnormal sensory phenomena (especially pain and paresthesias) which frequently complicate peripheral nerve injury in humans.     

The mechanisms of the recovery of nerve ending functions during the reinnervation of skeletal muscle

At the experiments on the frog cutaneous-pectoris muscle the nerve terminal functions in course of reinnervation process were investigated by electrophysiological and morphological methods. At the 20-25th days after the nerve crushing the nerve terminal response, which reflect the nerve terminal currents, formed the propagated action potential, were restored and the arising of evoked transmitter secretion occurred. The regenerating terminals are characterized by a low amplitude and altered shape of responses, by small velocity of the excitation propagation and the low level of evoked transmitter release. The 4-aminopyridine effect at the new formed nerve terminals was quite another, than at the intact nerve terminals. This data concluded, that the membrane of regenerating nerve terminal have a low density and a small gradient of sodium channels along the terminal and have not a calcium-activated potassium channels. It was proposed, that different kinds of channels are inserted into the nerve endings membrane at the different periods of the reinnervation process.     

Chronic immobilization stress reduces sodium intake and renal excretion in rats

The influence of chronic exposure to immobilization (IMO) on sodium appetite as well as sodium and potassium renal excretion in adult male Wistar rats was studied. The animals were individually housed and all variables under observation were measured in metabolic cages the first, seventh, and thirteenth days once the experiment had started. Half of the rats had access to water, and the remainder of the rats had access to both water and saline solution (1.5% NaCl). IMO reduced the intake of saline solution. Renal water, sodium, and potassium excretion in those IMO rats having access to saline were lower than in control rats. The effects of IMO were very similar during all observation days; therefore no evidence of adaptation to repeated stress was found. The present data indicate the following: (i) IMO stress reduced sodium appetite, probably as a secondary effect to the deficit in sodium renal excretion; (ii) IMO caused antidiuresis and antikaliuresis, only in those rats taking saline solution; (iii) no adaptation to repeated IMO stress was found in any of the tested variables. The reduction of sodium appetite observed in stressed rats might be a homeostatic mechanism to maintain sodium balance after impairment of renal sodium excretion caused by stress.     

Effects of xipamide and triamterene - alone or in combination - on electrolyte excretion in the rat

The effects of X and T in different doses alone or in combination on renal sodium and potassium excretion were examined in conscious rats. In acute experiments, the combination of both diuretics produced an approximately additive effect on sodium excretion. Addition of T to X in different dose relations (X:T = 1:0.5, 1:1 or 1:1.5) prevented the X-induced potassium loss. These effects were more pronounced at the highest dose of T; the difference to the other combinations, however, was small. In chronic experiments, when the diuretics were administered daily for 13 days, both combinations (X:T = 1:1 and X:T = 1:1.5) resulted in a nearly equilibrated potassium balance at the end of this period. In shorter intervals, however, sometimes considerable oscillations in potassium excretion occured.     

Calcium fluxes, ion currents and dihydropyridine receptors in a new immortal cell line from rat heart muscle

A cell line (RCVC) in permanent culture was developed from adult rat ventricular cells; transformation was attained by incubation with conditioned media from UCHT1, a rat thyroid cell line. Immortalized ventricular cells have a doubling time of 20 h, contact inhibition of growth, and display some muscle markers such as a high glycogen content and positive immunoreaction for myoglobin, alpha-sarcomeric actin, alpha-actinin and desmin. A microsomal fraction from these cells was shown to bind 3H-nitrendipine with a maximal capacity of 295 fmol/mg protein and an equilibrium dissociation constant of 0.7 nM. Nifedipine-sensitive 45Ca2+ influx was evident in partially depolarized cells (40 mM K+ in the incubation medium). An equivalent influx, induced by the calcium channel agonist BAYK-8644 and CGP-28392, was obtained in normally polarized cells. Patch clamp studies show slow inward currents that can be completely blocked by 5 microM nifedipine; cells were induced to further differentiation by culturing in a hormone supplemented medium for 30 days. Under this condition, fast, inactivating inward currents and a large outward current became apparent. After 40-60 days, the cells exhibit La(3+)-sensitive fast and slow inactivating inward currents that resemble T and L-type Ca2+ currents. This cell line appears to be a good model system for the investigation of cardiomyocyte differentiation in situ.     

Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations. A transient fast inactivating potassium current was activated at the most negative membrane potentials and was not reduced in the presence of 10 mM tetraethylammonium in the external solution. 4-Aminopyridine (2 mM) caused an 80% inhibition of this current. The activation of the fast inactivating potassium current was properly described by fitting a single exponent raised to the fourth power. The time constant of activation changed from 4 to 1 ms in the voltage range between -30 and +40 mV. The time constant of inactivation decreased from 35 to 15 ms over the same range of potentials. Parameters for the fit of a Boltzmann equation to mean values for steady-state activation were V1/2=-20mV, k=11.8mV, and for steady-state inactivation V1/2= -85 mV, k=-9.8 mV. A transient slow inactivating potassium current had an activation threshold between -40 and -30 mV. At 2 mM 4-aminopyridine, the depression of the slow potassium current was 55%. The extracellular application of 10 mM tetraethylammonium was less effective and evoked a 40% reduction. The activation of the slow inactivating potassium current was also described by a single exponential function raised to the fourth power. The time constant of activation decreased from 12 ms at a membrane potential of -10 mV to 4 ms at the potential of 60 mV. The inactivation of slow inactivating potassium current was described by two exponents. The time constant for the fast exponent ranged from 300 ms at -20 mV to 160 ms at +60 mV. The slower exponent was also potential dependent and its time constant ranged from approximately 2600 to 1600 ms over the same potentials. Parameters for the Boltzmann equation fittings to mean values were V1/2= -12.8 mV, k=13.4 mV and V1/2= -54.6 mV, k= -12 mV for steady-state activation and inactivation, respectively. A non-inactivating delayed rectifier potassium current was activated at the most positive membrane potentials. This non-inactivating current did not change in the presence of 4-aminopyridine. Extracellular tetraethylammonium (10 mM) caused a 70% reduction of this current. The activation of the non-inactivating potassium current was described by one exponent raised to the fourth power. The time constant for activation ranged from 85 ms at -5 mV to 30 ms at 45 mV. No time-dependent inactivation was observed during 15-s testing potentials in the voltage range between 10 and +60 mV. The activation behavior was characterized by V1/2=15.3 mV, k=12.5 mV. The densities of these potassium currents were studied for three groups of animals: one, five to six and 14-15 days of postnatal development. Fifty cells were examined in each age group. All three types of potassium currents were found in each investigated neuron. The mean densities of slow and fast inactivating potassium currents increased during ontogenetic development. The densities of non-inactivating delayed rectifier potassium current decreased in the first week of ontogenetic development and did not change thereafter.     

Oxygen sensing in airway chemoreceptors

Pulmonary neuroepithelial bodies, composed of innervated clusters of amine- and peptide-containing cells, are widely distributed throughout the airway mucosa of human and animal lungs. Structurally, neuroepithelial bodies resemble chemoreceptors (such as carotid body, taste buds) and are thought to function as hypoxia sensitive airway sensors. Evidence for this is indirect, however, and the mechanism of oxygen sensing by these cells is unknown. Here we culture neuroepithelial bodies isolated from rabbit fetal lungs and identify voltage-activated potassium, calcium and sodium currents using the whole-cell patch clamp technique. Upon exposure to hypoxia there is a reversible reduction (25-30%) in the outward potassium current, with no change in inward currents. In addition, we demonstrate the expression of an oxygen-binding protein (b-cytochrome, NADPH oxidase) on the plasma membrane of these cells. The identification of an oxygen-sensing mechanism (namely the presence of an O2-sensitive potassium channel coupled to an O2 sensor protein) in the cells of pulmonary neuroepithelial bodies indicates that they are transducers of the hypoxia stimulus and hence may function as airway chemoreceptors in the regulation of respiration.