G-protein dependent dual modulation by adenosine of ATP-evoked inward currents and intracellular Ca increase in PC12 cells.

α–latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G–protein–coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin–G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with Gα q/11 and Gα o but not with Gα s , Gα i or Gα z , indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX–evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca 2+ , LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca 2+ –dependent component of the toxin–evoked release. Based on (i) the known involvement of Gα q in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca 2+ in LTX action, we tested the effect of inhibitors of Ca 2+ mobilization on the toxin–evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca 2+ –dependent toxin's action. Thapsigargin, which depletes intracellular Ca 2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca 2+ . On the other hand, clostridial neurotoxins or drugs interfering with Ca 2+ metabolism do not inhibit the Ca 2+ –independent component of LTX–stimulated release. In the absence of Ca 2+ , the toxin induces in the presynaptic membrane non–selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca 2+ provided intracellular Ca 2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca 2+ , which then triggers secretion.

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Effects of ATP antagonists on purinoceptor-operated inward currents in PC12 cells

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)49631-5 ◽

1992 ◽

Vol 58 ◽

pp. 379

Author(s):

Kazuhide Inoue ◽

Ken Nakazawa ◽

Kannosuke Fujimori ◽

Norio Akaike ◽

Akira Takanaka

Keyword(s):

Pc12 Cells ◽

Inward Currents

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G proteins activate ionic conductances at multiple sites in T84 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.4.c1222 ◽

1997 ◽

Vol 272 (4) ◽

pp. C1222-C1231

Author(s):

L. Izu ◽

M. Li ◽

R. DeMuro ◽

M. E. Duffey

Keyword(s):

G Protein ◽

G Proteins ◽

Cell Voltage ◽

Equilibrium Potential ◽

T84 Cells ◽

Ionic Conductances ◽

Inward Currents ◽

Cl Channels ◽

K Current

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K+ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K+ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated large oscillating K+ and inward currents. This occurred even when Ca2+ was absent from the bath but not when the Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K+ was replaced with Na+ and cells were voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to pipettes activated large oscillating K+ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2+.

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Requirement of Pyk2 for the activation of the MAP kinase cascade induced by Ca2+ (but not by PKC or G protein) in PC12 cells

FEBS Letters ◽

10.1016/s0014-5793(99)01468-4 ◽

1999 ◽

Vol 461 (3) ◽

pp. 273-276 ◽

Cited By ~ 10

Author(s):

Rico Barsacchi ◽

Harald Heider ◽

Jean-Antoine Girault ◽

Jacopo Meldolesi

Keyword(s):

Map Kinase ◽

G Protein ◽

Pc12 Cells ◽

Map Kinase Cascade ◽

Kinase Cascade

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Vasopressin-Induced Currents in Rat Neonatal Spinal Lateral Horn Neurons Are G-Protein Mediated and Involve Two Conductances

Journal of Neurophysiology ◽

10.1152/jn.1998.80.4.1900 ◽

1998 ◽

Vol 80 (4) ◽

pp. 1900-1910 ◽

Cited By ~ 18

Author(s):

Miloslav Kolaj ◽

Leo P. Renaud

Keyword(s):

Spinal Cord ◽

G Protein ◽

Wide Distribution ◽

Channel Blockers ◽

Patch Clamp Recording ◽

Inward Current ◽

Lateral Column ◽

Lateral Horn ◽

Inward Currents ◽

Induced Currents

Kolaj, Miloslav and Leo P. Renaud. Vasopressin-induced currents in rat neonatal spinal lateral horn neurons are G-protein mediated and involve two conductances . J. Neurophysiol. 80: 1900–1910, 1998. Arginine vasopressin (AVP) receptors are expressed early in the developing spinal cord. To characterize AVP-induced conductances in lower thoracic sympathetic preganglionic (SPN) and other lateral horn neurons, we used patch-clamp recording techniques in neonatal (11–21 days) rat spinal cord slices. Most (90%) of 273 neurons, including all 68 SPNs, responded to AVP with membrane depolarization and/or a V1 receptor-mediated, dose-dependent (0.01–1.0 μM) and tetrodotoxin (TTX)-resistant inward current. A role for G-proteins was indicated by persistence of this inward current after intracellular dialysis with GTP-γ-S or GMP-PNP, its marked reduction with GDP-β-S, and significant reduction, but not abolition, after preincubation with pertussis toxin or in the presence of N-ethylmaleimide. Analysis of individual current-voltage ( I- V) relationships in 57 cells indicated the presence of two different membrane conductances. In 21 cells, net AVP-induced currents reversed around −103 mV, reflecting reduction in one or more barium-sensitive potassium conductances; in 12 cells, net AVP-induced current reversed around −40 mV and was not significantly sensitive to several potassium channel blockers including barium, tetraethylammonium chloride (TEA), 4-aminopyridine (4AP), cesium, or glibenclamide, suggesting increase in a nonselective cationic conductance that was separate from I h; in 24 cells where I- V lines shifted in parallel, AVP-induced inward currents were significantly greater and probably involved both conductances. These data indicate that SPNs and a majority of unidentified neonatal lateral horn neurons possess functional G-protein–coupled V1-type vasopressin receptors. The wide distribution of AVP receptors in neonatal spinal lateral column cells suggests a role that may extend beyond involvement in regulation of autonomic nervous system function.

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Human dipeptidyl peptidase III regulates G-protein coupled receptor-dependent Ca2+ concentration in human embryonic kidney 293T cells

Biological Chemistry ◽

10.1515/hsz-2016-0117 ◽

2016 ◽

Vol 397 (6) ◽

pp. 563-569 ◽

Cited By ~ 3

Author(s):

Subhash C. Prajapati ◽

Ratnakar Singh ◽

Shyam S. Chauhan

Keyword(s):

Angiotensin Ii ◽

G Protein ◽

Biological Function ◽

Dipeptidyl Peptidase ◽

Luciferase Reporter ◽

G Protein Coupled Receptor ◽

Intracellular Ca ◽

First Time ◽

G Protein Coupled

Abstract The precise biological function of human dipeptidyl peptidase III (hDPP III) is poorly understood. Using luciferase reporter constructs responsive to change in Ca2+ and/or cAMP and Fura 2-AM fluorometric assay, we show a significant decrease in intracellular Ca2+ following hDPP III overexpression and angiotensin II stimulation in angiotensin II type 1 receptor (G-protein coupled receptor, GPCR) expressing HEK293T cells. Silencing the expression of hDPP III by siRNA reversed the effect of hDPP III overexpression with a concomitant increase in Ca2+. These results, for the first time, show involvement of hDPP III in GPCR dependent Ca2+ regulation in HEK293T cells.

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Astrocyte-induced modulation of synaptic transmission

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y99-076 ◽

1999 ◽

Vol 77 (9) ◽

pp. 699-706 ◽

Cited By ~ 100

Author(s):

Alfonso Araque ◽

Rita P Sanzgiri ◽

Vladimir Parpura ◽

Philip G Haydon

Keyword(s):

Synaptic Transmission ◽

Glutamate Receptors ◽

Neuronal Activity ◽

Hippocampal Neurons ◽

Calcium Waves ◽

Metabotropic Glutamate ◽

Intracellular Ca ◽

Inward Currents ◽

Neuronal Electrical Activity ◽

Cultured Hippocampal Neurons

The idea that astrocytes simply provide structural and trophic support to neurons has been challenged by recent evidence demonstrating that astrocytes exhibit a form of excitability and communication based on intracellular Ca2+ variations and intercellular Ca2+ waves, which can be initiated by neuronal activity. These astrocyte Ca2+ variations have now been shown to induce glutamate-dependent Ca2+ elevations and slow inward currents in neurons. More recently, it has been demonstrated that synaptic transmission between cultured hippocampal neurons can be directly modulated by astrocytes. We have reported that astrocyte stimulation can increase the frequency of miniature synaptic currents. Furthermore, we also have demonstrated that an elevation in the intracellular Ca2+ in astrocytes induces a reduction in both excitatory and inhibitory evoked synaptic transmission through the activation of selective presynaptic metabotropic glutamate receptors.Key words: astrocyte-neuron signaling, glutamate receptors, calcium waves, neuronal electrical activity, synaptic transmission.

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Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

AJP Cell Physiology ◽

10.1152/ajpcell.00360.2014 ◽

2015 ◽

Vol 308 (8) ◽

pp. C608-C620 ◽

Cited By ~ 45

Author(s):

Mei Hong Zhu ◽

Tae Sik Sung ◽

Kate O'Driscoll ◽

Sang Don Koh ◽

Kenton M. Sanders

Keyword(s):

Slow Wave ◽

Interstitial Cells Of Cajal ◽

Ryanodine Receptors ◽

Interstitial Cells ◽

Pacemaker Activity ◽

Current Clamp ◽

Anoctamin 1 ◽

Intracellular Ca ◽

Inward Currents ◽

Cells Of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca2+-activated Cl− channels. We investigated the hypothesis that the Ca2+ responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca2+ stores. ICC, obtained from the small intestine of Kit +/copGFP mice, were studied under voltage and current clamp to determine the effects of blocking Ca2+ uptake into stores and release of Ca2+ via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca2+ concentration, suggesting that pacemaker activity depends on Ca2+ dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca2+ from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

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G protein βγ subunits interact with αβ- and γ-tubulin and play a role in microtubule assembly in PC12 cells

Cell Motility and the Cytoskeleton ◽

10.1002/cm.20234 ◽

2007 ◽

Vol 64 (12) ◽

pp. 936-950 ◽

Cited By ~ 13

Author(s):

Valentina Montoya ◽

Christina Gutierrez ◽

Omar Najera ◽

Denisse Leony ◽

Armando Varela-Ramirez ◽

...

Keyword(s):

G Protein ◽

Pc12 Cells ◽

Microtubule Assembly

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Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.277.5.g967 ◽

1999 ◽

Vol 277 (5) ◽

pp. G967-G975 ◽

Cited By ~ 9

Author(s):

Geoffrey I. Sandle ◽

Geoffrey Warhurst ◽

Ian Butterfield ◽

Norman B. Higgs ◽

Richard B. Lomax

Keyword(s):

G Protein ◽

Basolateral Membrane ◽

Secretory Process ◽

Synthetic Analog ◽

Channel Activity ◽

Patch Clamp Recording ◽

Channel Inhibition ◽

Colonic Crypts ◽

Intracellular Ca ◽

Dependent Mechanism

Somatostatin is a powerful inhibitor of intestinal Cl− secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K+ channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl− secretory process. Somatostatin (2 μM) elicited a >80% decrease in “spontaneous” K+ channel activity in cell-attached patches in nonstimulated crypts (50% inhibition =∼8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100–200 μM dibutyryl cAMP, 2 μM somatostatin and its synthetic analog octreotide (2 μM) both produced similar degrees of K+ channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl− (5 mM) conditions. In addition, 2 μM somatostatin abolished the increase in K+ channel activity stimulated by 2 μM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca2+. These results indicate that somatostatin peptides inhibit 23-pS basolateral K+ channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca2+sensitivity.

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