Possible mechanisms underlying effects of N-uronoyl derivatives of amino acids on electrical activity in snail neurons

Using intracellular recording, we studied the effects of N-uronoyl derivatives of an amino acid and peptides (1,2:3,4-di-O-isopropylidene-αa-D-galactopyranuronoyl)-β-alanine (DAGU-Ala), DAGU-glycyl-glycine (DAGU-Gly-Gly), DAGU-glycyl-D,L-glutamic acid (DAGU-Gly-Glu), as well as of 1,2:3,4-di-O-isopropylidene-αa-D-galactopyranosyluronic acid (DAGU itself), β-alanine (β-Ala), D,L-glutamic acid (D,L-Glu), and glycyl-glycine (Gly-Gly), which were added to the extracellular milieu, on the electrical activity of PPa1 and PPa2 neurons and unidentified neurons of Helix albescens Rossm. DAGU-Gly-Gly applied in concentrations of 10⁻⁴ to 10⁻² M hyperpolarized the membrane in a dose-dependent manner and decreased insignificantly the amplitude of action potentials (APs). Applications of DAGU-Ala, β-Ala, DAGU-Gly-Glu, D,L-Glu, and Gly-Gly in the same doses resulted in a shift of the membrane potential toward depolarization and in a drop in the amplitude of APs. Measurements of the first AP derivatives showed that all the above-mentioned substances suppressed in a concentration-dependent manner both inward and outward transmembrane ion currents. In this case, DAGU suppressed both inward and outward currents, while DAGU-Ala, β-Ala, DAGU-Glu, D,L-Glu, and Gly-Gly inhibited predominantly the outward potassium ion current; DAGU-Gly-Gly inhibited inward sodium and potassium ion currents. Results of a comparative analysis of the neurotropic action of the tested amino acids and their N-uronoyl derivatives showed that modification of the molecules of neurotransmitter amino acids leads to a decrease in their neurotoxicity and to an increase in their membranotropic properties. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 416–425, September–December, 2006.     

Components of depolarization-induced transmembrane ion current in isolated smooth muscle cells of the guinea pigtaenia coli

Transmembrane ion currents in isolated single smooth muscle cells (SMC) from the guinea pigtaenia coli were investigated using a whole-cell mode of the patch-clamp technique. Currents induced by depolarizing shifts in the membrane potential from its holding level of −60 mV contained an initial inward phase (Ca²⁺ current), which in 30–40 msec was followed by an outward phase. It was shown that outward current was carried by K ions and consisted at least of three components: one Ca²⁺-independent K⁺ current of delayed rectifier (KV) and two Ca²⁺-dependent K⁺ currents. The latter can be further divided into the apamin-sensitive (SK) and charybdotoxin-sensitive (BK) currents. It was found that relative contributions of these three components in total outward current at 0 mV were 35–45%, 5–15%, and 45–55% for KV, SK, and BK currents, respectively. A potential-dependent current carried by Ci ions was also found. This Cl⁻ current had inward direction within the range of potentials below the chloride equilibrium potential (E _Cl) and outward direction above theE _Cl. The magnitude of Cl⁻ current was significantly lower than the magnitude of total K⁺ current.     

Synaptic inhibition of smooth muscles of the human colon: Effects of vitamin B6 and its derivatives

Spontaneous activity, which is manifested as slow depolarization waves and action potentials, is observed in most (81%) smooth muscles (SMs) of the circular layer of the human colon. Independently of the type of pathology, inhibitory junction potentials (IJPs) in SMs of various parts of the human colon are evoked by intramural stimulation; ranges of parameters of these potentials were comparable with those observed in muscle intestinal fragments isolated at a distance of several tens of centimeters from the zone of injury. In muscle strips (MSs) of such fragments, pyridoxal-5′-phosphate (PPh) applied in different concentrations caused suppression of IJPs: in the concentration of 1·10⁻⁸ to 1·10⁻⁴ M it decreased the amplitude, and in the concentrations of 1·10⁻⁵ to 1·10⁻⁴ M and 1·10⁻⁴ M, respectively, it decreased rates of the half-amplitude rise and decay of these potentials. Pyridoxal (1·10⁻⁴ M) and 4-pyridoxolic acid (1·10⁻⁴ M) also caused a drop in the amplitude of IJPs; however, these agents influenced this parameter to a lesser extent, as compared with the effect of 1·10⁻⁴ M PPh. Pyridoxine (1·10⁻⁴ M) and pyridoxamine (1·10⁻⁴ M) evoked no significant changes in the parameters of IJPs in MSs of the human colon. Our data allow us to hypothesize that the suppressing effect of PPh on IJPs is determined by the presence of a purinergic component present in non-adrenergic inhibition of SMs of the human colon. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 269–279, July–August, 2006.     

Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton’s reaction in urine

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton’s system. Maximum CL emission is registered in urine containing H₂O₂ (5·10⁻⁴ M), Fe²⁺ (5·10⁻⁵ M), EDTA (5·10⁻⁵ M), and chemical enhancer lucigenin (10⁻⁴ M) at pH 5.5 and 36°C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10⁻³ and 10⁻⁴ M: for ascorbic acid, 5.92 ± 0.04 and 24.05 ± 1.82 μmol·sec⁻¹; for uric acid, 1.60 ± 0.02 and 21.45 ± 0.97 μmol·sec⁻¹, respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0–10 sec), fast flash of CL (10–50 sec), and latent period (50–300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 8, pp. 1062–1065.     

Two types of P2x-purinoceptors in neurons of the guinea pig ileum submucous plexus

Effects of exogenous adenosine 5′-triphosphate (ATP) on dissociated guinea pig ileum submucous neurons were studied using a conventional whole-cell patch-clamp technique. With the holding potential of −50 mV, application of 50–1,000 μM ATP evoked an inward current (ATP-induced current) in most (90%) of the tested neurons (n-35). ATP-induced currents were observed regardless of whether or not guanosine 5′-triphosphate (GTP, 0.2 mM) and ATP (2 mM) were present in the intracellular solution, or GTP was replaced with equimolar concentration of guanosine 5′-O-3-thiotriphosphate (n-5). In 26 of 29 neurons studied, which responded to ATP, applications of 50–1,000 μM ATP induced slowly declining currents. ATP receptors did not appear to be completely desensitized during a long pulse (up to 4 min) of 200 μM ATP. Suramin (200 μM) accelerated an increase to peak of the current induced by 200 μM ATP without affecting the maximum response amplitude (n−4_. In about 10% of the neuronsn−3), 50 μM ATP evoked rapidly declining (about 1 sec) currents. Application of 100 μM α,β-Me-ATP to these neurons evoked similar responses. The above results suggest that submucous neurons express two specific subtypes of ionotropic P_2x-purinoceptors, which might be involved in distinct excitatory processes in these neurons.     

The influence of IAA on the uptake of potassium,calcium, magnesium,water absorption and growth in young maize seedlings

The influence of IAA in two concentrations (10⁻⁸M and 10⁻⁵M) on relations between growth, water absorption and cation uptake and accumulation was tested.IAA in a higher concentration retarded growth remarkably. First of all, potassium uptake and water absorption were significantly decreased while the uptake of divalent cations was affected later and less remarkably. 10⁻⁸ M IAA accelerated the growth rate slightly together with acceleration of water absorption and cation uptake. Presented at the International Symposium “Plant Growth Regulators” held on June 18 – 22, 1984 at Liblice, Czechoslovakia.     

二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流和延迟整流钾电流的影响

运用全细胞膜片钳技术研究二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流(IA和ID)和延迟整流钾电流(IK)的影响。结果发现二氧化硫衍生物剂量依赖性地增大钾通道的电导,电压依赖性地增大钾电流的幅度,且这种增大作用部分可逆。二氧化硫非常显著地使延迟整流钾电流的激活过程向超极化方向移动,使瞬间外向钾电流的失活过程向去极化方向移动。10μmol/L二氧化硫衍生物作用前后,延迟整流钾电流的半数激活电压分别是(20.3±2.1)mV和(15.0±1.5)mV;IA和ID的半数失活电压分别朝去极化方向移动了6mV和7.4mV。这些结果表明二氧化硫改变了钾通道的特性,改变了神经元的兴奋性。     

D-Tubocurarine-Sensitive Component of Calcium-Dependent Potassium Current in Guinea Pig <Emphasis Type="Italic">Taenia Coli</Emphasis> Myocytes

Using a patch-clamp technique in the whole-cell configuration, we studied transmembrane ion currents in isolated single smooth muscle cells of the guinea pig taenia coli. A depolarizing step shift of the membrane potential from −50 mV was accompanied by the appearance of an outward current. Application of d-tubocurarine (d-TK) or a nonselective blocker of voltage-dependent potassium channels, tetraethylammonium (TEA), led to a decrease in the outward current. Application of d-TK against the background of the action of TEA additionally decreased the outward current. Analysis of the current-voltage (I–V) relationships of the d-TK-sensitive current showed that this current is practically voltage-independent. At the same time, an inflection of the I–V curve of the potassium current within the segment of maximum activation of the voltage-dependent potassium current is indicative of the sensitivity of this current to the intracellular Ca²⁺ concentration. Therefore, the calcium-activated potassium current through small-conductance calcium-dependent potassium channels includes a d-TK-sensitive voltage-independent component. Using depolarizing shifts of the membrane potential, we observed high- and low-amplitude spontaneous outward currents (SOCs) in many studied cells, i.e., the effect of an increase in the conductance of calcium-dependent potassium channels as a result of periodic release of Ca²⁺ from the intracellular stores. Application of d-TK led to a decrease in the frequency of low-amplitude SOCs and exerted nearly no influence on the high-amplitude SOCs under study. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 271–277, May–June, 2005.     

Inhibitory effects of various L-type and T-type calcium antagonists on electrically generated, potassium-induced and carbachol-induced contractions of porcine detrusor muscle

The inhibitory effects of different calcium antagonists on contractions of isolated porcine detrusor muscle were investigated. Suppression of the maximum potassium-induced contraction and electrically generated contractions by nifedipine, verapamil and diltiazem were investigated. Furthermore, concentration–response curves of carbachol after pretreatment with the L-type antagonists nifedipine, verapamil, diltiazem, nimodipine and the T-type antagonist mibefradil at different concentrations were performed. Nifedipine significantly reduced the potassium-induced maximum contraction to 89, 60, 21, 8 and 4% (10⁻⁹–10⁻⁵ M). Verapamil and diltiazem significantly reduced it to 64, 30 and 5% (10⁻⁷–10⁻⁵ M) or 79, 27, 7 and 1% (10⁻⁷–10⁻⁴ M), respectively. Nifedipine, verapamil and diltiazem significantly reduced the electrically generated contraction to 55, 36, 34 and 25% (10⁻⁷–10⁻⁴ M), 71, 32 and 2% (10⁻⁶–10⁻⁴ M), 96, 78, 38 and 5% (10⁻⁷–10⁻⁴ M), respectively. pD2 values of nifedipine, verapamil and diltiazem amounted to 7.07, 5.56 and 5.40 and differed significantly. After pretreatment with nifedipine at 10⁻⁶ M, the concentration–response curve of carbachol was nearly suppressed. The effects of nimodipine, verapamil and diltiazem were smaller. Mibefradil caused only at 10⁻⁵ M a significant reduction. All investigated L-type calcium antagonists were strong inhibitors of the examined contractions. Nifedipine showed the biggest inhibitory effect.     

Axon transport of steroid hormones via spinal root fibers in old rats

Labelled steroid hormones,³H-hydrocortisone and¹⁴C-testosterone, being injected in the gray matter of theL ₅−L ₆ spinal cord segments were shown to be transported via ventral and dorsal root fibers (antero- and retrograde directions, respectively) of old (25 to 28 months) rats with a lower velocity than in adult young (6 to 11 months) animals. The averaged maximum velocities of axon transport (AT) through the ventral and dorsal roots were: for³H-hydrocortisone, 756±63 and 738±46 mm per day, and for¹⁴H-testosterone, 624±54 and 608±80 mm per day, respectively. Therefore, in old rats the AT velocities for³H-hydrocortisone and¹⁴C-testosterone were about four and seven-eight times lower than those in adult rats. In the course of anterograde, AT through the ventral roots in old rats the inclusion of³H-hydrocortisone is sharply suppressed (by more than an order of magnitude), as compared with than in adult animals. The doses of non-labelled steroid hormones within a 10⁻⁷ – 10⁻⁶ range, injected into the lumbar spinal segments, resulted in hyperpolarization of muscle fibers of themm. gastrocnemius anddeltoideus, but this phenomenon developed in old rats much later than in adult rats. It is obvious that AT of steroid hormones can be considered one of the mechanisms of their effects on the tissue of an organism, and this mechanism undergoes extremely intensive modifications with aging.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

Patch-clamp analysis of voltage- and ligand-activated whole-cell currents in freshly dissociated neurons of the locust optic lobe

Whole-cell patch-clamp recordings were obtained from 116 freshly dissociated neuronal somata from the optic lobe of adult locusts (Schistocerca gregaria). Prerequisites were a papain treatment and the directed transfer of somata to the recording chamber by dabbing. Of the recorded somata, 65 were from lamina and 51 from other optic lobe neurons. All somata supported voltage-activated outward currents and some (24% of optic lobe, 3% of lamina neurons) also fast inward currents. Most lamina neurons supported an outward current that activated (V _1/2=−8.5 mV) and inactivated rapidly and a sustained outward current. Some lamina and most optic lobe neurons expressed only a sustained outward current (V _1/2=−9.4 mV). GABA and histamine elicited inward currents at negative holding potentials. Most optic lobe (95%) but only 18% of lamina neurons showed a γ-aminobutyric acid (GABA) current, whereas a similar percentage of optic lobe (50%) and lamina neurons (67%) expressed a histamine current. Both currents reversed near the chloride equilibrium potential, were reversibly reduced by picrotoxin, and did not show rundown. Thus, they likely represent chloride currents mediated by ionotropic receptors. Our data indicate that the lamina neurons recorded mainly represent monopolar cells postsynaptic to histaminergic photoreceptors. The optic lobe neurons, on which GABA and histamine apparently act as inhibitory neurotransmitters, are more heterogeneous. Accepted: 30 November 1997     

Regulation of Sorus Formation by Auxin in Laminariales Sporophyte

Young sporophytes of Laminaria japonica Areshoug were cultured in six indole-acetic acid (IAA) concentrations (0, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴ M) to examine the effect of auxin on growth. The effects of auxin on sorus formation were also examined by using discs taken from the adult sporophyte. The auxin contents and IAA oxidase activities in the thallus and sorus parts of the sporophyte were determined with the blade and sporophyll of other Laminariales plants, Undaria pinnatifida (Harvey) Suringar and Alaria crassifolia Kjellman. The young sporophytes of L. japonica showed highest elongation rate in 10⁻⁵ M IAA. In contrast, the sorus formation on the discs cultured in 10⁻⁵ M IAA was markedly delayed in comparison with other concentrations, indicating that sorus formation was suppressed by IAA. Free and conjugated auxin contents were lower in the reproductive parts than in the vegetative parts. In three Laminariales sporophytes, IAA oxidase activity was about 3–9 times higher in the reproductive parts than in the vegetative parts. Taken together these results suggest that the growth and reproduction of Laminariales sporophytes are regulated by internal auxin levels. Elucidating the regulation mechanism is likely to provide information that is important for the management of plant production and the assessment of the physiological status of plants in the field.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Isoflurane blocks glutamatergic excitatory transmission pre- and postsynaptically in crayfish muscle

Crayfish neuromuscular junctions are good models for the α-amino-hydroxy-5-methyl-4-isoxazol-propionic acid-type of vertebrate brain excitatory synapses. The action of a typical volatile anaesthetic, isoflurane, was studied on the excitatory postsynaptic currents recorded with a perfused macropatch electrode. Isoflurane reduced quantal exitatory postsynaptic currents in amplitude, in their rise time and in the decay time constant. Small such effects were elicited by <1 mmol · l⁻¹ isoflurane, while the maximal isoflurane concentration of 7 mmol · l⁻¹ reduced the amplitude to about a quarter and shortened the decay time constant even more, while the rise time was diminished by about a quarter. This combination of effects is typical for an open channel block for which an approximate binding rate constant of isoflurane of 6 · 10⁵ mol⁻¹l · s⁻¹ and an unbinding rate of 10–100 s⁻¹ is derived. In addition to this postsynaptic effect, isoflurane inhibited the release of transmitter quanta from the terminal, for instance with 2.5 mmol · l⁻¹ isoflurane by a factor of 7.3 ± 6.3 (SD). In the glutamatergic nerve terminals release is modulated by low glutamate concentrations via a metabotropic autoreceptor which is blocked by the combination of 6-cyano-7-nitro-quinoxaline-2,3-dione and dl-2-amino-5-phosphor-valeric acid. This blocker combination also can prevent the inhibition of release by isoflurane, and it may be suggested that isoflurane elicits inhibition of release through the metabotropic presynaptic glutamate receptors. Accepted: 29 March 1998     

Effect of peroxynitrite on production of superoxide anion radicals and calcium homeostasis in cells of astroglial origin

We studied the effect of a donor of peroxynitrite, SIN-1, on the morphological characteristics of interweaved rat C6 glioma cells, on menadione-induced production of superoxide anion radicals, and on the concentration of Ca²⁺ in these cells. In concentrations of 1.25·10⁻⁴ to 2.5·10⁻⁷ M, SIN-1 demonstrated cytotoxic and antimitogenic effects. This donor of peroxynitrite caused abnormal modifications of the size of C6 cells and the structure of cellular organelles, intensified in a dose-dependent manner the release of Ca²⁺ from cellular stores into the cytoplasm, and suppressed menadione-induced production of superoxide anion radicals. Therefore, it should be believed that peroxynitrite exerts a modifying effect on the processes of mitotic division and induces apoptosis; it is also involved in the processes of intracellular signalling providing an increase in the concentration of cytosolic Ca²⁺ and a decrease in the redox activity of cells. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 401–406, September–December, 2006.     

Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)

A protocol was developed for plant regeneration of Melia azedarach L. by in vitro culture of apical meristem (0.5 mm in length). The influence of six clones was investigated. The culture procedure comprised two sequential steps: 1) Induction of shoots by in vitro culture of axillary buds from adult trees (10–15 years old) by culture on Murashige and Skoog (1962) medium (MS) supplemented with 0.5 mg·dm⁻³ BAP (6-benzylaminopurine), 0.1 mg·dm⁻³ IBA (indolebutyric acid), and 0.1 mg·dm⁻³ GA₃ (gibberellic acid). The Multiplication of the regenerated shoots was achieved in MS + 0.5 mg·dm⁻³ BAP + 0.1 mg·dm⁻³ GA₃. 2) In vitro culture of the apical meristems from the regenerated shoots in MS medium (0.7 %) supplemented with various combinations of BAP and IBA. Maximum shoot proliferation was obtained on MS medium supplemented with 0.5 mg·dm⁻³ BAP and 0.1 mg·dm⁻³ IBA. Regenerated shoots were rooted on MS + 3.5 mg·dm⁻³ IBA (4 days) followed by subculture on MS lacking growth regulators (30 days). Complete plants were transferred to soil.     

Voltage-activated potassium currents in the somatic membrane of sensory neurons of early postnatal rats

Transmembrane ion currents were studied in the somatic membrane of freshly isolated neurons from the spinal ganglia of early postnatal (younger than 15-day-old) rats. According to their dissimilar voltage dependence and different sensitivity to external application of tetraethylammonium (TEA) and 4-aminopyridine (4-AP), three types of outward potassium currents were identified. Fast-inactivating K⁺ current was activated at the most negative values of the membrane potential and showed the highest sensitivity to external application of 4-AP. The threshold for activation of slow-inactivating K⁺ current was within a −40 ... −30 mV range. Non-inactivating delay-rectified current showed the highest sensitivity to TEA. All three types of K⁺ currents could be found in all studied neurons of animals of three age groups: 1, 5 to 6, and 14 to 15 postnatal days. The mean density of fast-inactivating K⁺ current significantly increased during the first two weeks of postnatal ontogenesis. Within the studied period, the mode of a normal (Gaussian) distribution of fast K⁺ current shifted toward higher current density values. The mean density of slow-inactivating K⁺ current also increased with the age. Yet, the mean density of non-inactivating delay-rectified K⁺ current significantly dropped during the first five days of the postnatal development and remained stable during the following time interval.     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997