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 Inhibition of L-type calcium-channel activity by thapsigargin and 2,5-t-butylhydroquinone, but not by cyclopiazonic acid

1994 ◽  
Vol 302 (1) ◽  
pp. 147-154 ◽  
Author(s):  
E J Nelson ◽  
C C R Li ◽  
R Bangalore ◽  
T Benson ◽  
R S Kass ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Cyclopiazonic Acid ◽  
Calcium Currents ◽  
Pituitary Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Low Concentrations ◽  
Ic50 Values

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.

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.

Protein kinase activator 1-oleoyl-2-acetyl-sn-glycerol inhibits two types of calcium currents in GH3 cells

1988 ◽  
Vol 254 (1) ◽  
pp. C206-C210 ◽  
Author(s):  
C. Marchetti ◽  
A. M. Brown
Keyword(s):  
Protein Kinase ◽  
Chick Embryo ◽  
Calcium Currents ◽  
Gh3 Cells ◽  
High Threshold ◽  
Pituitary Cells ◽  
Excitable Cells ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Low Threshold

Two types of Ca2+ currents, high-threshold, long-lasting, or L currents and low-threshold, transient, or T currents, are present in many excitable cells. L-type Ca2+ current is modulated by, among others, beta- and alpha-adrenoreceptors and intracellular Ca2+, but modulation of T-type Ca2+ current is less well established. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic activator of protein kinase C (PKC), modulates whole cell Ca2+ currents in a variety of excitable cells. Whether activators of PKC affect preferentially L and T types of Ca2+ currents is unknown. We tested OAGs effects on whole cell Ca2+ currents in the clonal GH3 line of anterior pituitary cells. The currents were measured using the whole cell patch-clamp method. Four to 60 microM OAG reversibly reduced Ca2+ currents produced by test potentials to 10 mV, and the inhibition was half maximal at approximately 25 microM. Such concentrations depress Ca2+ currents in chick embryo dorsal root ganglion (DRG) cells and clonal AtT-20 pituitary cells. To test whether OAG acted preferentially on L or T current, we separated the two using depolarizing prepulses to inactivate T current. OAG (40 microM) attenuated T currents by 60% and L currents by 50%. The current waveforms were not changed and were simply scaled, and the effects on both occurred approximately 15 s after OAG was applied. In chick embryo DRGs OAG inhibited the T current by 30% and the L current by 50%. We conclude that PKC modulates Ca2+ currents by acting on both L and T Ca2+ channels.

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.

Effects of cannabinoids on endogenous K+ and Ca2+ currents in HEK293 cells

2003 ◽  
Vol 81 (5) ◽  
pp. 436-442 ◽  
Author(s):  
Clemente Vásquez ◽  
Ricardo A Navarro-Polanco ◽  
Miguel Huerta ◽  
Xóchitl Trujillo ◽  
Felipa Andrade ◽  
...  
Keyword(s):  
Cannabinoid Receptor ◽  
Inward Rectifier ◽  
Calcium Currents ◽  
Hek293 Cells ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Inverse Agonists ◽  
Cb1 Cannabinoid Receptor ◽  
Cell Variant ◽  
Cannabinoid Agonists

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 μM) decreased the calcium current by 53.1 ± 2.6, 47.5 ± 1.2, and 38.8 ± 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 ± 3.6, 83.7 ± 5.1, and 63.0 ± 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.Key words: cannabinoids, WIN 55,212-2, anandamide, methanandamide, inverse agonists.

Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells

1992 ◽  
Vol 262 (5) ◽  
pp. C1211-C1219 ◽  
Author(s):  
A. A. Haymes ◽  
Y. W. Kwan ◽  
J. P. Arena ◽  
R. S. Kass ◽  
P. M. Hinkle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ion Concentration ◽  
Free Calcium ◽  
Pituitary Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Channel Current ◽  
Kinase C

These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca2+ channels of GH3 pituitary cells. The rate of 45Ca2+ uptake was stimulated greater than 25-fold by depolarization in the presence of BAY K 8644; the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced this response by 70% in a concentration-dependent fashion. Phorbol 12,13-dibutyrate (PDBu) inhibited depolarization-induced 45Ca2+ uptake within 1 min and caused a nearly maximal reduction after 1 h; its effects were rapidly reversible. TPA decreased the high K(+)-stimulated increase in intracellular free calcium ion concentration ([Ca2+]i) from 8.5- to 3.2-fold by 5 min and to 2.0-fold after 18 h without altering the peak [Ca2+]i response to the peptide hormone TRH. Ca2+ channel current, measured directly using the whole cell configuration of the patch-clamp technique, declined an average of 6.4% over 5 min for control cells and 28.9% when TPA was added to the bathing medium for 5 min. Treatment with 100 nM TPA for 24 h dramatically reduced peak current without shifting the peak of the current-voltage relationship. The mean peak Ca2+ channel current was reduced from 423 to 128 pA, although a few cells seemed completely resistant. To determine whether the effects of phorbol esters were due to the activation of PKC we tested the potency of several drugs to inhibit L-channel activity and to shift the affinity of the epidermal growth factor (EGF) receptor, an established PKC response.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals Calcium currents in embryonic and neonatal mammalian skeletal muscle.

1988 ◽  
Vol 91 (6) ◽  
pp. 781-798 ◽  
Author(s):  
K G Beam ◽  
C M Knudson
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Primary Cultures ◽  
Ionic Currents ◽  
Cell Types ◽  
Calcium Currents ◽  
Mammalian Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Complete Inactivation ◽  
Inward Currents

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.

Calcium Currents in Solitary Hair Cells Isolated from Frog Crista Ampullaris

1992 ◽  
Vol 2 (1) ◽  
pp. 31-39 ◽  
Author(s):  
I. Prigioni ◽  
S. Masetto ◽  
G. Russo ◽  
V. Taglietti
Keyword(s):  
Hair Cells ◽  
Semicircular Canals ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Extracellular Solution ◽  
Homogeneous Population ◽  
Average Value ◽  
Crista Ampullaris ◽  
Basal Pole ◽  
Inward Currents

Some properties of Ca2+ currents in hair cells isolated from frog semicircular canals by enzymatic or mechanical treatment were studied by using the whole-cell configuration of the patch-clamp technique. After blocking the large outward K+ currents by substituting Cs+ for K+ and adding tetraethylammonium to the pipette filling solution, voltage- and time-dependent inward currents were clearly detectable in the presence of 4 mM Ca2+ in the extracellular solution. Ca2+ current was recruited at test potentials more positive than -60 mV, showed a rapid activation, and exhibited no inactivation during 150-ms depolarizing pulses. The maximal amplitude was attained at about -20 mV, with an average value of about 80 pA. When Ca2+ in the extracellular solution was replaced with Ba2+, the magnitude of inward currents increased about twofold. Ba2+ currents were blocked more effectively by Cd2+ than by Ni2+, were suppressed by 0.5 μM ω-conotoxin, and were virtually unaffected by amiloride. The dihydropyridine Bay K 8644 caused a marked voltage-dependent increase in inward currents. The present data suggest that hair cells from frog crista ampullaris are endowed with a homogeneous population of Ca2+ channels having several properties similar to those described for neuronal L channels. Since these channels are recruited in a range of potentials close to the resting level, it is suggested that they subserve the control of both resting and evoked transmitter release from the basal pole of the hair cells.

scholarly journals Bay K 8644 reveals two components of L-type Ca2+ channel current in clonal rat pituitary cells.

1996 ◽  
Vol 108 (1) ◽  
pp. 1-11 ◽  
Author(s):  
D M Fass ◽  
E S Levitan
Keyword(s):  
Voltage Dependence ◽  
Slow Component ◽  
Fast Component ◽  
Bay K 8644 ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Independent Manner ◽  
Channel Current ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

Whole-cell L-type Ca2+ channel current was recorded in GH3 clonal rat pituitary cells using Ba2+ as a charge carrier. In the presence of the dihydropyridine agonist Bay K 8644, deactivation was best described by two exponential components with time constants of approximately 2 and approximately 8 ms when recorded at -40 mV. The slow component activated at more negative potentials than the fast component: Half-maximal activation for the slow and fast components occurred at approximately -15 and approximately 1 mV, respectively. The fast component was more sensitive to enhancement by racemic Bay K 8644 than the slow component: ED50fast = approximately 21 nM, ED50slow = approximately 74 nM. Thyrotropin-releasing hormone (TRH; 1 microM) inhibited the slow component by approximately 46%, whereas the fast component was inhibited by approximately 22%. TRH inhibition of total L-current showed some voltage dependence, but each Bay K 8644-revealed component of L-current was inhibited in a voltage-independent manner. Therefore, the apparent voltage dependence of TRH action is derived from complexities in channel gating rather than from relief of inhibition at high voltages. In summary, Bay K 8644-enhanced L-currents in GH3 cells consist of two components with different sensitivities to voltage, racemic Bay K 8644, and the neuropeptide TRH.

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