Membrane Hyperpolarization In Response Research Articles

Effect of Alternating Electric Current on Pulsation Mode of Rat Major Arteries In Situ.

In experiments on narcotized rats, the electrical potential and impedance of isolated segment of the right femoral and/or carotid artery were simultaneously recorded in situ via two extracellular nonpolarizable Ag/AgCl electrodes mounted along the arteries at the interelectrode distance of 4 mm. The active, passive, and intermediate pulsing modes of arterial segment were determined according to the phase relations between its electrical impedance and BP, which was simultaneously measured in the symmetrical part of the respective left artery and used to assess pressure in the examined segment. The study assessed the effect of amplitude (0.2-2.0 mA) of alternating probe current (100 kHz), which was used to measure the electrical impedance of arterial segment, on its pulsing mode. The pulsing mode determined at the initial minimal probe current of 0.2 mA was passive with out-of-phase pulsatile oscillations of electrical impedance and BP. After elevation of the probe current amplitude to maximal level of 2 mA, these oscillations became in-phase indicating transition of the arterial segment to active pulsing mode. This transition was accompanied by appearance of arterial voltage impulses synchronized with BP upstrokes and an 11-fold median increase in the peak-to-peak value of electrical impedance oscillations with the interdecile range of 7-15 (N=28). Under moderate amplitude of probe current (0.3-0.5 mA), the intermediate mode of arterial pulsing was observed featured by a delayed, weak, and short active constriction during BP front, which was insufficient to resist and counterbalance the dilating effect of rising BP. In this case, the pulsatile oscillations of electrical impedance were smaller than those observed in active or passive pulsing modes indicating a possibility to stabilize the arterial diameter during pulsatile oscillations of BP. The effect of alternating electric current on the mode of arterial pulsation is explained with electrical model of smooth muscle cell membrane reflecting the rectifying features of potassium channels and predicting membrane hyperpolarization in response to external alternating current passing across the cell. The visibilities of therapeutic neurotropic and angiotropic stimulation with alternating electric current are discussed.

Reversal of dopamine-mediated firing inhibition through activation of the dopamine transporter in substantia nigra pars compacta neurons.

One of the hallmarks of ventral midbrain dopamine-releasing neurons is membrane hyperpolarization in response to stimulation of somato-dendritic D2 receptors. At early postnatal age, under sustained dopamine, this inhibitory response is followed by a slow recovery, resulting in dopamine inhibition reversal (DIR). In the present investigation, we aimed to get a better insight into the cellular mechanisms underlying DIR. We performed single-unit extracellular recordings with a multi-electrode array device and conventional patch-clamp recordings on midbrain mouse slices. While continuous dopamine (100μM) perfusion gave rise to firing inhibition that recovered in 10 to 15min, the same effect was not obtained with the D2 receptor agonist quinpirole (100nM). Moreover, firing inhibition caused by the GABAB receptor agonist baclofen (300nM) was reversed by dopamine (100μM), albeit D2 receptors had been blocked by sulpiride (10μM). Conversely, the block of the dopamine transporter (DAT) with cocaine (30μM) prevented firing recovery by dopamine under GABAB receptor stimulation. Accordingly, in whole-cell recordings from single cells, the baclofen-induced outward current was counteracted by dopamine (100μM) in the presence of sulpiride (10μM), and this effect was prevented by the DAT antagonists cocaine (30μM) and GBR12909 (2μM). Our results indicate that the DAT plays a major role in DIR, mediating it under conditions of sustained dopamine exposure, and point to DAT as an important target for pharmacological therapies leading to prolonged enhancement of the dopaminergic signal.

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca2+-activated K+ channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca2+ and voltage-gated potassium (BKCa) channels. Here, we explored the effect of LPI on intermediate-conductance Ca2+-activated K+ (IKCa) channels using excised inside-out patches from endothelial cells. The functional expression of IKCa was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IKCa channels with a slope conductance of 39 pS in symmetric K+ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IKCa single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IKCa single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.

The GPR55 agonist lysophosphatidylinositol acts as an intracellular messenger and bidirectionally modulates Ca2+-activated large-conductance K+ channels in endothelial cells

Lysophospholipids are known to serve as intra- and extracellular messengers affecting many physiological processes. Lysophosphatidylinositol (LPI), which is produced in endothelial cells, acts as an endogenous agonist of the orphan receptor, G protein-coupled receptor 55 (GPR55). Stimulation of GPR55 by LPI evokes an intracellular Ca2+ rise in several cell types including endothelial cells. In this study, we investigated additional direct, receptor-independent effects of LPI on endothelial large-conductance Ca2+ and voltage-gated potassium (BKCa) channels. Electrophysiological experiments in the inside-out configuration revealed that LPI directly affects the BKCa channel gating properties. This effect of LPI strictly depended on the presence of Ca2+ and was concentration-dependent, reversible, and dual in nature. The modulating effects of LPI on endothelial BKCa channels correlated with their initial open probability (Po): stimulation at low Po (<0.3) and inhibition at high Po levels (>0.3). In the whole-cell configuration, LPI in the pipette facilitated membrane hyperpolarization in response to low (0.1–2 μM) histamine concentrations. In contrast, LPI counteracted membrane hyperpolarization in response to supramaximal cell stimulation with histamine. These results highlight a novel receptor-independent and direct bidirectional modulation of BKCa channels by LPI on endothelial cells. We conclude that LPI via this mechanism serves as an important modulator of endothelial electrical responses to cell stimulation.

Photosensitive Signal Transduction Induces Membrane Hyperpolarization in Paramecium bursaria

The protozoan ciliate Paramecium bursaria exhibits membrane hyperpolarization in response to photostimulation, accompanied with an increased swimming speed. The external addition of cyclic nucleotide phosphodiesterase (PDE) inhibitors, either theophylline (1,3-dimethylxanthine) or 3-isobutyl-1-methylxanthin (IBMX), increased in both amplitudes of the membrane hyperpolarization and the increase in swimming speed. Moreover, the addition of membrane permeable cyclic nucleotide analogs, either 8-bromo-adenosine 3',5'-cyclic monophosphate (Br-cAMP) or 8-Br-guanosine 3',5'-cyclic monophosphate (Br-cGMP), increased these amplitudes. On the other hand, the addition of l-cis-diltiazem, known to block the conductance of cyclic nucleotide-gated channels, partially decreased both amplitudes of the membrane hyperpolarization and the increase in swimming speed. An enzyme immunoassay of cellular cyclic nucleotide contents showed that photostimulation induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP), but little increase in guanosine 3',5'-cyclic monophosphate (cGMP), raising the possibility that a rapid increase in cAMP mediates the light-induced hyperpolarization in P. bursaria.

Reduced function of endothelial prostacyclin in human omental resistance arteries in pre-eclampsia.

It remains unclear in pre-eclampsia whether or not a functional change occurs in the role played by prostacyclin in endothelium-dependent relaxation in resistance arteries. We examined this using human omental resistance arteries (obtained from pre-eclamptic or normotensive pregnant women) in the presence of N(G)-nitro-L-arginine (L-NNA, an inhibitor of nitric oxide synthase). In endothelium-intact strips from both groups, 9,11-epithio-11,12-methano-thromboxane A(2) (STA(2), a thromboxane A(2) mimetic) produced a contraction. Diclofenac (an inhibitor of cyclooxygenase) enhanced the STA(2) contraction only in the normotensive pregnant group (1.4 times control, P < 0.01). In the presence of STA(2), bradykinin (0.1 microM) produced an endothelium-dependent relaxation in both groups, the relaxation being significantly smaller for the pre-eclamptic group (P < 0.002). Diclofenac significantly attenuated the bradykinin-induced relaxation only for the normotensive pregnant group (31 % inhibition, P < 0.001). The bradykinin-induced membrane hyperpolarization consisted of diclofenac-sensitive and -insensitive components. The former, but not the latter, was significantly smaller in pre-eclampsia (-4.3 vs. -2.6 mV, P < 0.05). The concentrations of 6-keto-PGF(1alpha) (a stable metabolite of prostacyclin) in these arteries were significantly lower in pre-eclampsia in both the absence and presence of bradykinin (about 0.2-0.4 times the normotensive pregnant value in each case, P < 0.01). By contrast, both the relaxation and the membrane hyperpolarization in response to beraprost (10 nM, a stable analogue of prostacyclin) were similar between the two groups. We conclude that, in pre-eclampsia, a reduced part is played by prostaglandins in the endothelium-dependent relaxation seen in resistance arteries and that this may be due to a reduced production of prostacyclin by the endothelium.

Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells.

In guard cells, membrane hyperpolarization in response to a blue light (BL) stimulus is achieved by the activation of a plasma membrane H(+)-ATPase. Using the patch clamp technique on broad bean (Vicia faba) guard cells we demonstrate that both steady-state- and BL-induced pump currents require ATP and are blocked by vanadate perfused into the guard cell during patch clamp recording. Background-pump current and BL-activated currents are voltage independent over a wide range of membrane potentials. During BL-activated responses significant hyperpolarization is achieved that is sufficient to promote K(+) uptake. BL activation of pump current becomes desensitized by three or four pulses of 30 s x 100 micromol m(-2) s(-1) BL. This desensitization is not a result of pump inhibition as maximal responses to fusicoccin are observed after full BL desensitization. BL treatments prior to whole cell recording show that BL desensitization is not due to washout of a secondary messenger by whole cell perfusion, but appears to be an important feature of the BL-stimulated pump response. We found no evidence for an electrogenic BL-stimulated redox chain in the plasma membrane of guard cells as no steady-state- or BL-activated currents are detected with NADH or NADPH added to the cytosol in the absence of ATP. Steady-state- nor BL-activated currents are affected by the inclusion along with ATP of 1 mM NADH in the pipette under saturating red light or by including NADPH in the pipette under darkness or saturating red light. These data suggest that reduced products of photosynthesis do not significantly modulate plasma membrane pump currents and are unlikely to be critical regulators in BL-stimulation of the plasma membrane H(+)-ATPase in guard cells.

Origin and function of epoxyeicosatrienoic acids in vascular endothelial cells: more than just endothelium-derived hyperpolarizing factor?

1. In addition to their contribution to endothelium-derived hyperpolarization, our understanding of the physiological function of epoxyeicosatrienoic acids (EET) within the vascular wall and the actual enzymes involved in the formation of the EET in endothelial cells is very limited. In the present study, the expression of potential cytochrome P450 (CYP) mono/epoxygenases was assessed in endothelial cells isolated from porcine and bovine aortas as well as in the human umbilical vein-derived cell lines EA.hy926 and ECV304. 2. Expression of CYP2B1, CYP2E1 and CYP3A could be found. The latter were inducible by dexamethasone/clofibrate for 72 h, a procedure that also enhanced CYP epoxygenase activity in endothelial cells. 3. Enzyme induction yielded increases in capacitative Ca2+ entry and membrane hyperpolarization in response to autacoids, such as bradykinin and thapsigargin. Thiopentone sodium, an inhibitor of endothelial CYP mono/epoxygenase(s), diminished autacoid-induced capacitative Ca2+ entry and membrane hyperpolarization, while the effect of EET remained unchanged. 4. Epoxyeicosatrienoic acids activated endothelial tyrosine kinase activity in a concentration-dependent manner. Arachidonic acid, at 20-fold higher concentrations, also increased tyrosine kinase activity. Because only the effect of arachidonic acid was inhibited by thiopentone sodium, an inhibitor of CYP mono/epoxygenases, these data suggest that arachidonic acid needs to be converted to the EET in order to stimulate tyrosine kinase. 5. All these data provide clear evidence that the CYP epoxygenase-derived arachidonic acid metabolites (EET) not only serve as potential endothelium-derived hyperpolarizing factors but also constitute highly active intracellular messengers with a physiological role including the control of Ca2+ signalling, membrane potential and tyrosine kinase activity.

Distribution of chemoreceptors to quinine on the cell surface of Paramecium caudatum

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni2+-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.

Transmembrane potential responses during HL-60 promyelocyte differentiation.

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/ macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC2-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by KCa channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl- conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions.

Inwardly rectifying potassium conductance can accelerate the hyperpolarizing response in retinal horizontal cells.

1. We studied the activation properties and assessed the functional role of the inwardly rectifying potassium conductance (GK.IR) in acutely isolated retinal horizontal cells (HCs) with the use of the whole cell patch-clamp technique. 2. The potassium current mediated by GK.IR was isolated by the use of Cs+ or Ba2+ ions. This current was outward, although relatively small in amplitude, in the voltage range between the potassium equilibrium potential (EK) and 50-60 mV more positive. The current reversed its polarity at EK and became inward at potentials more negative than EK. When HCs were bathed in normal Ringer (EK = -90 mV), GK.IR began to active at about -30 mV, was 30-40% activated at the resting potential (-70 to -80 mV) and about fully activated at -130 mV. Thus a significant portion of the activation range of GK.IR overlaps the HC physiological response range (-20 to -80 mV). 3. GK.IR has a dramatic effect on the kinetics of membrane polarization. Blocking GK.IR with Cs+ or Ba2+ significantly slowed the rate of membrane hyperpolarization in response to a hyperpolarizing current ramp over the HC physiological response range. Blocking GK.IR also dramatically slowed the onset rate of a simulated light response generated by a brief break in a sustained glutamate puff. 4. These results suggest that GK.IR can enhance the temporal resolution of the HC by accelerating the onset rate of the hyperpolarizing light response.

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons). Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.

Variation in the pattern of [Ca2+]i change induced by acetylcholine in cultured hippocampal neurons

Acetylcholine (ACh) caused various patterns of change in the intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal neurons. We studied the underlying mechanisms of the [Ca2+]i changes with simultaneous recording of [Ca2+]i and membrane potential/current. In most cases, [Ca2+]i rise was accompanied by a membrane depolarization. The [Ca2+]i change was significantly reduced when the membrane was voltage clamped, which implies that most of the [Ca2+]i rise results from the Ca2+ influx through the voltage-gated Ca2+ channel activated by the membrane depolarization. The membrane depolarizations were classified into two types, one associated with membrane conductance decrease and the other associated with membrane conductance increase. The former results from potassium conductance ((gK+) decrease, and the latter may result from the activation of a Na(+)-permeable channel. However, [Ca2+]i elevation was also observed in some neurons showing membrane hyperpolarization in response to ACh. This seems to show that ACh liberates Ca2+ from the intracellular Ca2+ store, resulting in the activation of a calcium-dependent K+ channel (KCa). The variations of ACh response in the hippocampal neurons seem to result from a variety of muscarinic acetylcholine receptors and various species of ion channels governed by those receptors.

Actions of gamma-aminobutyric acid on rat supraoptic nucleus neurosecretory neurones in vitro.

1. Intracellular recordings were obtained from thirty-eight rat supraoptic nucleus (s.o.n.) neurosecretory neurones in perfused hypothalamic explants. Changes in membrane potential and conductance were monitored following application of gamma-aminobutyric acid (GABA), and related agonists and antagonists. 2. GABA depressed action potential discharge of all of thirty-five s.o.n. neurones tested and induced either membrane hyperpolarization or depolarization. Neurones that displayed membrane hyperpolarization in response to lower GABA concentrations (30-300 microM) demonstrated a biphasic membrane voltage change with a later depolarizing phase as a response to higher concentrations (up to 3000 microM). 3. GABA (10-3000 microM) induced a prominent concentration-dependent increase in membrane conductance in all neurones. The critical slope for the log-log plot of [GABA] vs. GABA-induced membrane conductance was 1.7, indicating co-operativity in the GABA receptor-induced conductance change. 4. Muscimol (0.3-30 microM) potently mimicked all the effects of GABA. Bicuculline (1-100 microM) antagonized the effects of GABA and muscimol in a competitive manner. 5. Glycine and taurine (1-10 mM) had weak effects, although comparatively similar to those of GABA. These actions were blocked both by bicuculline (100 microM) and by strychnine (1 microM). At higher concentrations (greater than 10 microM), strychnine also antagonized the actions of GABA. 6. In recordings with potassium-acetate-filled micropipettes, the reversal potential of hyperpolarizing membrane voltage responses to GABA was -72.5 +/- 1.5 mV in close agreement (+/- 5 mV) with the reversal potential of inhibitory post-synaptic potentials (i.p.s.p.s) recorded in the same neurones. Depolarizing responses to GABA reversed polarity at -50 +/- 1.6 mV. In recordings with KCl-filled micropipettes, voltage responses to GABA were always depolarizing and reversed near -40.0 +/- 4.3 mV. Similarly, reduction of the concentration of chloride ions in the perfusion medium from 134 to 10.4 mM induced a positive shift of the GABA reversal potential by 40-50 mV. 7. From measurements of input resistance (Rin) and cell time constant (tau O), input capacitance (Cin; representing total membrane capacitance) was calculated as 78.9 +/- 2.1 pF. During responses to GABA or muscimol, decreased Rin was accompanied by a linearly related decrease in tau o indicating that these substances had no effect on the membrane capacitance of s.o.n. neurones.(ABSTRACT TRUNCATED AT 400 WORDS)