Two-pore domain K channel, TASK-1, in pulmonary artery smooth muscle cells.

Pulmonary vascular tone is strongly influenced by the resting membrane potential of smooth muscle cells, depolarization promoting Ca2+ influx, and contraction. The resting potential is determined largely by the activity of K+-selective ion channels, the molecular nature of which has been debated for some time. In this study, we provide strong evidence that the two-pore domain K+ channel, TASK-1, mediates a noninactivating, background K+ current (IKN), which sets the resting membrane potential in rabbit pulmonary artery smooth muscle cells (PASMCs). TASK-1 mRNA was found to be present in PASMCs, and the membranes of PASMCs contained TASK-1 protein. Both IKN and the resting potential were found to be exquisitely sensitive to extracellular pH, acidosis inhibiting the current and causing depolarization. Moreover, IKN and the resting potential were enhanced by halothane (1 mmol/L), inhibited by Zn2+ (100 to 200 micromol/L) and anandamide (10 micromol/L), but insensitive to cytoplasmic Ca2+. These properties are all diagnostic of TASK-1 channels and add to previously identified features of IKN that are shared with TASK-1, such as inhibition by hypoxia, low sensitivity to 4-aminopyridine and quinine and insensitivity to tetraethylammonium ions. It is therefore concluded that TASK-1 channels are major contributors to the resting potential in pulmonary artery smooth muscle. They are likely to play an important role in mediating pulmonary vascular responses to changes in extracellular pH, and they could be responsible for the modulatory effects of pH on hypoxic pulmonary vasoconstriction.

The effect of acetylcholine and serotonin on calcium transients and calcium currents in identified Helix pomatia L. neurons.

The results presented demonstrate that in D neurons of the snail Helix pomatia L., acetylcholine (ACh) (10 divided by 100 microM) and serotonin (5-HT) (0.1 divided by 1000 microM) applications reduce both the basal intracellular concentration level ([Ca2+]in) and the amplitudes of calcium transients induced by membrane depolarization. It is likely that the mechanism of [Ca2+]in changes in the suppression of calcium inward currents (ICa). Influences of Ach and 5-HT on ICa were studied. Both effects were dose-dependent (ACh--0.01 divided by 100 microM and 5-HT--0.1 divided by 1000 microM). The half-maximal effects (IC50) were evoked by ACh concentration of 0.15 microM and 5-HT--15 microM. Furthermore we have also shown that in some cells 5-HT could evoke a transient increase in ICa (IC50 = 2 microM). The effects of Ach and 5-HT were nonadditive--the subsequent application of ACh after 5-HT, and vice versa, produced no inhibitory effects. This may indicate that both substances act through a common intermediate (possibly, G-protein).

Dopamine blocks T-type calcium channels in cultured rat adrenal glomerulosa cells.

Dopamine is known to inhibit aldosterone secretion. In the present study using whole-cell voltage-clamp technique we found that dopamine, bromocriptine and quinpirole inhibit low-threshold (T-type) voltage dependent Ca2+ channels. The inhibiton was sustained and reversible, and it was prevented by sulpiride. These findings indicate that the effect of dopamine was mediated via DA2 receptors.

Inhibition of an identified snail postsynaptic neuron evoked by stimulation of a peptidergic interneuron initiating bursting pacemaker activity in another neuron.

Beating neuron V8, whose activity is abolished upon interneuronal stimulation, has been found and identified in the CNS of the snail, Helix pomatia. It was found earlier that stimulation of the same interneuron initiates bursting pacemaker activity in another neuron. Under voltage clamp conditions interneuronal stimulation evoked a slow outward current in the V8 neuron with a latency of about 2 s. Intracellular injection of tetraethylammonium or Cs+ ions into the interneuron greatly increased the slow outward current amplitude. External application of CdCl2 (1 mM), LaCl3 (5 mM) or replacement of Ca2+ by Mg2+ ions reversibly abolished transmission between the interneuron and the V8 neuron. The neuronal membrane conductance increased by about three times on the maximum of the slow outward current development. Computer fitting showed that a decay of the slow outward current can be approximated by the sum of two exponentially decaying components with time constants of about 1 s and 16 s. Extrapolated reversal potential for a fast (1 s) component of the current was about -70 mV. A slow (16 s) component asymptotically decreased upon hyperpolarization and it was observed at even more negative potential than Ek. Theophylline (1 mM) and 3-isobutyl-1-methylxanthine (0.1 mM) reversibly increased the slow outward current amplitude, whereas imidazole (5 mM) and tolbutamide (5 mM) caused its reversible decrease. It is concluded that inhibitory monosynaptic transmission exists between the interneuron and the beating V8 neuron. Interneuronal simulation evokes an increase in the membrane potassium conductance and probably a decrease in stationary potential-dependent sodium conductance of the postsynaptic V8 neuron. The cellular cyclase system seems to be involved in the postsynaptic response.

Lead ions close steady-state sodium channels in Helix neurons.

Extracellularly applied Pb2+ (1-150 microM) induced an outward current (IPb) in intracellularly perfused snail neurons. The current-voltage relationship of the Pb(2+)-induced current was linear over the potential range of -100 to -40 mV with negative slope conductance. The Pb-induced current was strongly dependent on the Na+ gradient. The IPb in intra- or extracellular K+- and Cl(-)-free or -rich solutions was almost the same as in control external and internal salines. The negative slope of the I-V curve and the decreased conductivity during Pb2+ application suggested that IPb is owing to the blocking of the resting Na conductance. Data obtained from single-channel measurements also supported this conclusion. Patch-clamp data showed that the steady-state Na channel has a conductance of 14 pS and both closed and open time-distributions displayed single exponential character.

Neurochemical, electrophysiological and immunocytochemical evidence for a noradrenergic link between the sympathetic nervous system and thymocytes.

The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.

Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2-sensing potassium current.

1. The contributions of specific K+ currents to the resting membrane potential of rabbit isolated, pulmonary artery myocytes, and their modulation by hypoxia, were investigated by use of the whole-cell, patch-clamp technique. 2. In the presence of 10 microM glibenclamide the resting potential (-50 +/- 4 mV, n = 18) was unaffected by 10 microM tetraethylammonium ions, 200 nM charybdotoxin, 200 nM iberiotoxin, 100 microM ouabain or 100 microM digitoxin. The negative potential was therefore maintained without ATP-sensitive (KATP) or large conductance Ca(2+)-sensitive (BKCa) K channels, and without the Na(+)-K+ ATPase. 3. The resting potential, the delayed rectifier current (IK(V)) and the A-like K+ current (IK(A)) were all reduced in a concentration-dependent manner by 4-aminopyridine (4-AP) and by quinine. 4. 4-AP was equally potent at reducing the resting potential and IK(V), 10 mM causing depolarization from -44 mV to -22 mV with accompanying inhibition of IK(V) by 56% and IK(A) by 79%. In marked contrast, the effects of quinine on resting potential were poorly correlated with its effects on both IK(A) and IK(V). At 10 mM, quinine reduced IK(V) and IK(A) by 47% and 38%, respectively, with no change in the resting potential. At 100 microM, both currents were almost abolished while the resting potential was reduced < 50%. Raising the concentration to 1 mM had little further effect on IK(A) or IK(V), but essentially abolished the resting potential. 5. Reduction of the resting potential by quinine was correlated with inhibition of a voltage-gated, low threshold, non-inactivating K+ current, IK(N). Thus, 100 microM quinine reduced both IK(N) and the resting potential by around 50%. 6. The resting membrane potential was the same whether measured after clamping the cell at -80 mV, or immediately after a prolonged period of depolarization at 0 mV, which inactivated IK(A) and IK(V), but not IK(N). 7. When exposed to a hypoxic solution, the O2 tension near the cell fell from 125 +/- 6 to 14 +/- 2 mmHg (n = 20), resulting in a slow depolarization of the myocyte membrane to -35 +/- 3 mV (n = 16). The depolarization occurred without a change in the amplitude of IK(V) or IK(A), but it was accompanied by 60% inhibition of IK(N) at 0 mV. 8. Our findings suggest that the resting potential of rabbit pulmonary artery myocytes depends on IK(N), and that inhibition of IK(N) may mediate the depolarization induced by hypoxia.

Influence of chronic hypoxia on the contributions of non-inactivating and delayed rectifier K currents to the resting potential and tone of rat pulmonary artery smooth muscle.

Exposing rats to chronic hypoxia increased the 4-aminopyridine (4-AP) sensitivity of pulmonary arteries. 1 mM 4-AP caused smooth muscle cell depolarization and contraction in arteries from hypoxic rats, but had little effect in age-matched controls. Chronic hypoxia downregulated delayed rectifier K+ current (IK(V)), which was nearly 50% blocked by 1 mM 4-AP, and non-inactivating K+ current (IK(N)), which was little affected by 1 mM 4-AP. The results suggest that IK(N) determines resting potential in control rats and that its downregulation following hypoxia leads to depolarization, which activates IK(V) and increases its contribution to resting potential. The hypoxia-induced increase in 4-AP sensitivity thus reflects a switch in the major K+ current determining resting potential, from IK(N) to IK(V). This has important implications for the actions and specificity of pulmonary vasodilator drugs.

Neurohypophysial peptides selectively depressed high voltage-activated Ca-current in snail neurons.

It was found that neurohypophysial peptides oxytocin and Lys-vasopressin selectively decreased the high voltage-activated component of the inward Ca-current in voltage-clamped Helix pomatia identified neurons in a dose- and time-dependent manner. This effect was unaffected or further enhanced applying phosphodiesterases inhibitors. It is suggested that suppression of high voltage-activated current was due to the activation of the adenylate cyclase system, likely protein kinase A.

The effect of alpha-latrotoxin on a synaptic connection between identified neurons in the brain of the mollusc Helix pomatia L.

The effect of alpha-latrotoxin on identified monosynaptic peptidergic contacts between identified neurons from the brain of the snail Helix pomatia L. was studied. It was found that, after extracellular application, toxin evoked an increase in the amplitude of the postsynaptic response. Neither amplitude nor duration of the action potential in a presynaptic neuron was affected. Intracellular injection of toxin into the soma of a presynaptic neuron led to a decrease in the postsynaptic current amplitude. The current induced by intracellular injection of cAMP into a postsynaptic neuron was also inhibited by extracellular or intracellular application of toxin. These data indicate that toxin evokes both an increase of transmitter release from a presynaptic neuron and a decrease in amplitude of the postsynaptic response.

Influence of oxytocin on identified neurons of the brain of the snail Helix pomatia L.

The responses of identified neurons induced by the effect of perfusion with a solution of oxytocin were investigated in this study. Depolarizing, hyperpolarizing, and modulating types of responses were found. It is hypothesized that these responses are associated in the majority of instances with the system of cyclic nucleotides.

Voltage-clamp and single channel analysis of Pb(2+)-induced current in isolated snail neurons.

Pb-activated outward current was investigated in intracellularly perfused, isolated snail neurons. Pb-ions induced non-inactivating but reversible current (IPb). The IPb showed concentration dependence. The I-V curve was linear with negative slope and the IPb proved to be Na-dependent.

[The effect of oxytocin on the identified neurons of the brain in the edible snail Helix pomatia L]. / Vliianie oksitotsina na identifitsirovannye neirony mozga vinogradnoi ulitki Helix pomatia L.

Responses induced by a perfusion by a solution with oxytocin were examined in identified Helix pomatia L. neurons. Depolarizing, hyperpolarizing, and modulatory neuronal responses were observed. The responses under study were supposed to be associated in most of the cases with the system of cyclic nucleotides.

[Effect on an isolated "bursting" snail neuron of an endogenous peptide initiating bursting activity]. / Vliianie na izolirovannyi "pachechnyi" neiron ulitki éndogennogo peptida, initsiruiushchego ritmovodiashchuiu aktivnost'.

Application of an endogenous peptide ("initiating factor") isolated from the ganglia of the snail Helix pomatia elicited bursting activity in a silent isolated "bursting" neuron. Oxytocin application under the same conditions produced only depolarization of the neuronal membrane.

[The effect of arginine vasopressin and its derivatives on the brain neurons of the edible snail]. / Vliianie arginin-vazopressina i ego proizvodnykh na neirony mozga vinogradnoi ulitki.

Neuronal responses induced by application or perfusion of solutions with arginine vasopressin and its derivatives (VPS) have been examined on identified Helix pomatia neurons. No differences between VPS-induced effects were obtained. Depolarizing, hyperpolarizing and modulatory neuronal responses were found. De- and hyperpolarizing responses were due to an increase and a decrease in permeability of the surface neuronal membrane, respectively. The reversal potential of both responses was near the chloride equilibrium potential and was inhibited by furosemide. It is supposed that VPS-induced responses are associated with an increase and a decrease in permeability of chloride ions of the surface neuronal membrane. A modulatory VPS response is an increase of the current amplitude induced by intracellular cAMP injection by VPS perfusion. It is supposed that some Helix pomatia neurons have different receptors to VPS which, probably, are associated with the system of cyclic nucleotides.

[Isolation of a peptide initiating burst activity in an identified neuron of Helix pomatia]. / Vydelenie peptida, initsiruiushchego pachechnuiu aktivnost', u identifitsirovannogo neirona vinogradnoi ulitki.

A peptide was isolated from the water-soluble fraction of brain extract of the snail (Helix pomatia). The peptide initiated or enhanced the bursting activity of the identified neuron RPa1. The effect of the peptide application was similar to the effect of the total water-soluble fraction. The action of the peptide on neuronal bursting activity was not identical to the action of other known peptides. It is suggested that under normal conditions the peptide is released from the presynaptic terminals of a nonidentified interneuron on the soma of RPa1 neuron initiating its bursting activity.

Effect of molluscan neuropeptide RAPYFVamide on identified Helix pomatia L. neurons.

1. The effect of Mytilus inhibitory peptide-related peptide RAPYFVamide, isolated from Helix pomatia brain, was studied on 21 different identified Helix neurons. 2. It was found that the neuropeptide hyperpolarized 11 of the 21 neurons studied, inducing a K-dependent current. Not all neurons responded by hyperpolarization to the peptide application, though, in all cases the voltage-dependent outward K and inward Ca currents were depressed. 3. This may show that, in the peptide effect, more than one type of K conductance is involved. 4. It is proposed that RAPYFVamide may have a functional role in the modulation of the feeding behavior in H. pomatia.

A study of the connection between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snail Helix pomatia.

The connection between an interneuron initiating pacemaker activity in the bursting RPa1 neuron and the bursting neuron itself (Pin and Gola, 1983) has been analyzed in the snail Helix pomatia. Prolonged depolarization of the interneuronal membrane produced in it a series of action potentials as well as a parallel initiation or enhancement of bursting activity in the RPa1 neuron. If the discharge in the interneuron was evoked by short current pulses of threshold amplitude, no bursting activity was seen in the RPa1 neuron. However, short stimuli delivered on the background of subthreshold depolarization of the interneuronal membrane produced bursting activity in the RPa1 neuron. Under voltage-clamp conditions a slow inward current could be recorded in the RPa1 neuronal membrane after stimulation of the interneuron with a latency of about 2 sec. Short shifts of the holding potential in the hyperpolarizing direction at the maximum of this current produced a transient outward current. Replacement of extracellular Ca2+ by Mg2+ ions, as well as addition of 1 mM CdCl2 to the external solution, prevented the response to the interneuronal stimulation in the RPa1 neuron. Electron microscopic investigation of the interneuron has shown the abundance of Golgi complexes in its cytoplasm with electron-dense granules in their vicinity. It is concluded that the connection between the interneuron and the bursting neuron is of chemical origin, based on secretion by the former of some substances which activate at least two types of ionic channels in the membrane of the RPa1 neuron.

[Postsynaptic mechanisms of the initiation of burst activity in neuron PPa1 of Helix pomatia in response to an interneuron]. / Postsinapticheskie mekhanizmy initsiatsii pachechnoi aktivnosti v neirone PPa1 vinogradnoi ulitki pod vliianiem interneirona.

Postsynaptic mechanisms of the connection between an interneuron initiating the bursting activity in neurons RPa1 and V7 and the neurons themselves were examined. Under voltage clamp conditions a slow inward current could be recorded in these neurons after stimulation of the interneuron. A decrease of temperature reduced the speed of rise and fall of this current with a temperature coefficient about 10. The current-voltage characteristic of the slow inward current had a maximum near -65 mV. The decrease in external concentration of Na+ ions led to a decrease of its amplitude. At the maximum of this current short hyperpolarizing pulses induced outward currents instead of inward ones. It is concluded that interneuronal stimulation activate at least two types of ionic channels in the bursting neurons soma. This process, perhaps, is mediated via a chain of cytoplasmic biochemical reactions.

[Monosynaptic pathway responsible for generation of burst activity in neuron PPa1 of Helix pomatia]. / Monosinapticheskii put', otvetstvennyi za generatsiiu pachechnoi aktivnosti v neirone PPa1 vinogradnoi ulitki.

The connections between an interneuron in the visceral ganglion initiating the pacemaker activity in RPa1 neuron and RPa1 neuron itself have been analyzed in snail. The interneuron stimulation led to initiation of the bursting activity in the RPa1 neuron. Replacement of extracellular Ca2+ by Mg2+ ions, as well as addition of CdCl2 to the external solution reversibly inhibited the effect of the interneuronal stimulation on the RPa1 neuron. An increase of extracellular Ca2+ ions concentration to 70 mM did not prevent the effect. Intracellular Cs+ and TEA injection in the interneuron increasing the action potential duration in the interneuron increased also the effectiveness of connection between the neurons. It is concluded that the connection between the neurons under investigation is monosynaptic and peptidergic.