Cloned δ-Opioid Receptors in GH3 Cells Inhibit Spontaneous Ca2+ Oscillations and Prolactin Release ThroughK IR Channel Activation

2000 ◽  
Vol 83 (5) ◽  
pp. 2691-2698 ◽  
Author(s):  
Elemer T. Piros ◽  
Rew C. Charles ◽  
Lei Song ◽  
Chris J. Evans ◽  
Tim G. Hales
Keyword(s):  
Opioid Receptors ◽  
Receptor Agonist ◽  
Phosphatidyl Inositol ◽  
Gh3 Cells ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Prolactin Release ◽  
Dependent Inhibition ◽  
Intracellular Ca ◽  
Inward Currents

Opioid receptors can couple to K+ and Ca2+ channels, adenylyl cyclase, and phosphatidyl inositol turnover. Any of these actions may be important in the regulation of neurotransmitter and hormone release from excitable cells. GH3 cells exhibit spontaneous oscillations of intracellular Ca2+concentration ([Ca2+]i) and prolactin release. Activation of cloned δ-opioid receptors stably expressed in GH3 cells inhibits both spontaneous Ca2+signaling and basal prolactin release. The objective of this study was to examine a possible role for K+ channels in these processes using the patch-clamp technique, fluorescence imaging, and a sensitive ELISA for prolactin. The selective δ receptor agonist [d-Pen2,d-Pen2]enkephalin (DPDPE) inhibited [Ca2+]i oscillations in GH3 cells expressing both μ and δ receptors (GH3MORDOR cells) but had no effect on control GH3 cells or cells expressing μ receptors alone (GH3MOR cells). The inhibition of [Ca2+]i oscillations by DPDPE was unaffected by thapsigargin pretreatment, suggesting that this effect is independent of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. DPDPE caused a concentration-dependent inhibition of prolactin release from GH3MORDOR cells with an IC50 of 4 nM. DPDPE increased inward K+current recorded from GH3MORDOR cells but had no significant effect on K+ currents recorded from control GH3 cells or GH3MOR cells. The μ receptor agonist morphine also had no effect on currents recorded from control cells but activated inward K+ currents recorded from GH3MOR and GH3MORDOR cells. Somatostatin activated inward currents recorded from all three cell lines. The DPDPE-sensitive K+ current was inwardly rectifying and was inhibited by Ba2+ but not TEA. DPDPE had no effect on delayed rectifier-, Ca2+-, and voltage-activated or A-type K+ currents, recorded from GH3MORDOR cells. Ba2+ attenuated the inhibition of [Ca2+]i and prolactin release by DPDPE, whereas TEA had no effect, consistent with an involvement of K IR channels in these actions of the opioid.

scholarly journals Properties of two types of calcium channels in clonal pituitary cells.

1986 ◽  
Vol 87 (1) ◽  
pp. 161-182 ◽  
Author(s):  
D R Matteson ◽  
C M Armstrong
Keyword(s):  
Calcium Currents ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Patch Clamp Technique ◽  
Barium Ions ◽  
Channel Current ◽  
Inward Currents ◽  
A Cell ◽  
Cell Variant ◽  
Sodium And Potassium

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.

scholarly journals Characterization of the Inhibitory Effect of Gastrodigenin and Gastrodin on M-type K+ Currents in Pituitary Cells and Hippocampal Neurons

2019 ◽  
Vol 21 (1) ◽  
pp. 117 ◽  
Author(s):  
Chih-Sheng Yang ◽  
Ming-Chi Lai ◽  
Ping-Yen Liu ◽  
Yi-Ching Lo ◽  
Chin-Wei Huang ◽  
...  
Keyword(s):  
Current Density ◽  
Time Course ◽  
Gh3 Cells ◽  
Delayed Rectifier ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Activation Time ◽  
Patch Clamp Technique ◽  
Type K ◽  
K Current

Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

2005 ◽  
Vol 289 (5) ◽  
pp. G935-G948 ◽  
Author(s):  
Toshio Ohta ◽  
Akane Kubota ◽  
Matsuka Murakami ◽  
Ken-ichi Otsuguro ◽  
Shigeo Ito
Keyword(s):  
Rank Order ◽  
Reversal Potential ◽  
Specific Protein ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Patch Clamp Technique ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
Inward Currents

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At −60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP ≥ adenosine 5′- O-3-triphosphate ≥ CTP ≥ 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

2000 ◽  
Vol 278 (4) ◽  
pp. G644-G651 ◽  
Author(s):  
M. Hanani ◽  
M. Francke ◽  
W. Härtig ◽  
J. Grosche ◽  
A. Reichenbach ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Glial Cells ◽  
Muscle Layer ◽  
Membrane Receptors ◽  
Delayed Rectifier ◽  
Longitudinal Muscle Layer ◽  
Inward Currents ◽  
Morphological Identity ◽  
Clamp Study ◽  
Myenteric Ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na+ and Ca2+currents as well as delayed-rectifier K+ currents. Immunohistochemical staining confirmed that most neurons have Na+ channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K+ inward currents and depolarization-induced K+ outward currents. Glia showed large “passive” currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.

scholarly journals Adenosine A1receptor-mediated antiadrenergic effects are modulated by A2a receptor activation in rat heart

1999 ◽  
Vol 276 (2) ◽  
pp. H341-H349 ◽  
Author(s):  
Gavin R. Norton ◽  
Angela J. Woodiwiss ◽  
Robert J. McGinn ◽  
Mojca Lorbar ◽  
Eugene S. Chung ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Receptor Agonist ◽  
Ventricular Myocytes ◽  
Physiological Role ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Receptor Antagonists ◽  
Rat Hearts ◽  
Intracellular Ca ◽  
Perfused Rat Hearts

Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10−8 M)-elicited contractile responses (+dP/d t max) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10−7 M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10−7 M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10−5 M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 × 10−7 M)-elicited increases in intracellular Ca2+concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]itransients in the absence of exogenous adenosine. These results indicate that adenosine A2areceptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.

scholarly journals Dependence of Spontaneous Electrical Activity and Basal Prolactin Release on Nonselective Cation Channels in Pituitary Lactotrophs

2012 ◽  
pp. 267-275 ◽  
Author(s):  
M. KUČKA ◽  
K. KRETSCHMANNOVÁ ◽  
S. S. STOJILKOVIC ◽  
H. ZEMKOVÁ ◽  
M. TOMIĆ
Keyword(s):  
Electrical Activity ◽  
Action Potentials ◽  
Gh3 Cells ◽  
Cation Channels ◽  
Organic Cations ◽  
Trpc Channels ◽  
Pituitary Cells ◽  
Prolactin Release ◽  
Nonselective Cation Channels ◽  
Mrna Transcripts

All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH3 cells, replacement of extracellular Na+ with large organic cations, but not blockade of voltage-gated Na+ influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at –50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na+ with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH3 cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH3 cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca2+ influx and prolactin release in a concentration-dependent manner. Gd3+, a common Ca2+ channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca2+ influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca2+ influx and hormone release in lactotrophs and GH3 cells.

scholarly journals The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

2008 ◽  
pp. 55-62
Author(s):  
HY Xu ◽  
X Huang ◽  
M Yang ◽  
J-B Sun ◽  
L-H Piao ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Phosphodiesterase Inhibitor ◽  
Potassium Currents ◽  
Inhibitory Effect ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein ◽  
Gastric Myocytes

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.

scholarly journals Effect of FMRFamide on voltage-dependent currents in identified centrifugal neurons of the optic lobe of the cuttlefish, Sepia officinalis

2020 ◽  
Author(s):  
Abdesslam Chrachri
Keyword(s):  
Visual Processing ◽  
Calcium Current ◽  
Optic Lobe ◽  
Low Voltage ◽  
Sodium Current ◽  
Calcium Currents ◽  
Delayed Rectifier ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents

AbstractWhole-cell patch-clamp recordings from identified centrifugal neurons of the optic lobe in a slice preparation allowed the characterization of five voltage-dependent currents; two outward and three inward currents. The outward currents were; the 4-aminopyridine-sensitive transient potassium or A-current (IA), the TEA-sensitive sustained current or delayed rectifier (IK). The inward currents were; the tetrodotoxin-sensitive transient current or sodium current (INa). The second is the cobalt- and cadmium-sensitive sustained current which is enhanced by barium and blocked by the dihydropyridine antagonist, nifedipine suggesting that it could be the L-type calcium current (ICaL). Finally, another transient inward current, also carried by calcium, but unlike the L-type, this current is activated at more negative potentials and resembles the low-voltage-activated or T-type calcium current (ICaT) of other preparations.Application of the neuropeptide FMRFamide caused a significant attenuation to the peak amplitude of both sodium and sustained calcium currents without any apparent effect on the transient calcium current. Furthermore, FMRFamide also caused a reduction of both outward currents in these centrifugal neurons. The fact that FMRFamide reduced the magnitude of four of five characterized currents could suggest that this neuropeptide may act as a strong inhibitory agent on these neurons.SummaryFMRFamide modulate the ionic currents in identified centrifugal neurons in the optic lobe of cuttlefish: thus, FMRFamide could play a key role in visual processing of these animals.

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