Alpha 1 adrenoceptor activation potentiates taurine response mediated by protein kinase C in substantia nigra neurons

1996 ◽  
Vol 76 (4) ◽  
pp. 2455-2460 ◽  
Author(s):  
J. Nabekura ◽  
T. Omura ◽  
N. Horimoto ◽  
T. Ogawa ◽  
N. Akaike
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substantia Nigra ◽  
Single Channel ◽  
Glycine Receptor ◽  
Peak Amplitude ◽  
Whole Cell ◽  
Kinase C ◽  
Channel Opening ◽  
S 100

1. The potentiation of glycine receptor-mediated taurine response (Itau) by alpha 1 adrenoceptor activation was investigated in neurons freshly dissociated from the rat substantia nigra (SN) using a nystatin perforated-patch recording. 2. Norepinephrine (NE) at a concentration of 10(-4) M in the presence of 10(-5) M yohimbine and 10(-5) M propranolol potentiated the peak amplitude of Itau (10(-3) M) at a holding potential of -40 mV under voltage clamp conditions. NE could be substituted by phenylephrine at this potentiation. 3. This potentiation of the taurine response persisted in the treatment with pertussis toxin (500 ng/ml) for 18 h. The intracellular application of GDP-beta S (100 microM) with a conventional whole cell patch recording mode abolished the effect of alpha 1 adrenoceptor activation on the Itau. 4. Staurosporine (10(-7) M) blocked the enhancement of Itau by 10(-4) M NE with 10(-5) M yohimbine and 10(-5) M propranolol. In additional phorbol-12-myristate 13-acetate (10(-5) M) potentiated Itau. 5. The intracellular application of 0.275 U/ml protein kinase C (PKC) with a conventional whole cell configuration gradually increased the peak amplitude of Itau. On the other hand, intracellular perfusion either without PKC or with PKC plus 4 microM PKC (19-36), a PKC inhibitor, did not potentiate Itau. 6. A single channel recording in a cell attached configuration revealed that NE (10(-4) M) with 10(-5) M yohimbine and 10(-5) M propranolol increased the total open time of the taurine-activated channel. This increase of the channel opening was antagonized by staurosporine (10(-7) M). 7. Neither tapsigargin (10(-6) M), LiCl (10(-4) M), trifluoperazine (10(-5) M) nor (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (10(-4) M) applied in the perfusate were found to affect the potentiation of Itau by alpha 1 adrenoceptor. The intracellular application of inositol triphosphates (10(-4) M) in a conventional whole cell recording also had no effect on Itau. 8. These findings thus indicate that alpha 1 adrenoceptor coupled with pertussis-insensitive G protein increases the intracellular PKC activity, thus leading to an increase in the channel opening activated by taurine and an enhancement of the peak amplitude of Itau in the SN neurons.

Abstract 174: Biophysical Basis of Protein Kinase C Inhibition of the Novel alpha1D Calcium Channel

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Mohamed Chahine ◽  
Yongxia Qu ◽  
Mohamed Boutjdir
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Channel ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Kinase C ◽  
Pkc Activation

The recently reported α 1D calcium channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single channel level is not known. Therefore, the effect of PKC activation was studied on α 1D calcium channel expressed in tsA201 cells using cell-attached method. Unitary currents were recorded in the presence of 70 mM Ba 2+ as the charge carrier. Unitary currents were evoked by 500 ms depolarizing pulses from a holding potential of −80 mV every 0.5 Hz. Under basal condition, channel activity was rare and infrequent, however Bay K 8644 (1 μM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by PMA (10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the α 1D calcium channel. This is consistent with a decreased entry of α 1D Ca channel into open states in the presence of PMA. These data show, for the fist time, 1) the α 1D calcium channel activity at the single channel level and 2) the biophysical basis of by which PKC activation inhibits the α 1D calcium channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the α 1D Ca-channel activity and the reduction in whole-cell α 1D Ca current previously reported. Altogether, these data are relevant to the understanding of the patho-physiology of α 1D calcium channel and its regulation by the autonomics.

Isoflurane-induced Facilitation of the Cardiac Sarcolemmal KATPChannel

2002 ◽  
Vol 97 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Kazuhiro Fujimoto ◽  
Zeljko J. Bosnjak ◽  
Wai-Meng Kwok
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Channel ◽  
Intact Cell ◽  
Direct Interaction ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
Kinase C ◽  
Inside Out

Background Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (K(ATP)) channels. However, evidence for a direct effect of volatile anesthetic on the K(ATP) channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal K(ATP) channel were investigated. Methods Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal K(ATP) channel current. Results In the cell-attached patch configuration, 2,4-dinitrophenol (150 microm) opened the sarcolemmal K(ATP) channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the K(ATP) channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the K(ATP) channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When K(ATP) current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the K(ATP) current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the K(ATP) channel activated by 12,13-dibutyrate in a cell-free environment. Conclusions Isoflurane facilitated the opening of the sarcolemmal K(ATP) channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the K(ATP) channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal K(ATP) channel may have a significant role in anesthetic-induced preconditioning.

Regulation of cation channels in liver cells by intracellular calcium and protein kinase C

1994 ◽  
Vol 266 (4) ◽  
pp. G677-G684 ◽  
Author(s):  
J. G. Fitz ◽  
A. H. Sostman ◽  
J. P. Middleton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Resting Potential ◽  
Cation Channels ◽  
Nucleotide Receptors ◽  
Direct Role ◽  
Intact Cells ◽  
Kinase C ◽  
Channel Opening ◽  
Gamma S

The regulation of Ca(2+)-permeant cation channels in HTC hepatoma cells was investigated using patch clamp and fluorescence techniques. In intact cells, exposure to nucleotide analogues ATP, uridine 5'-triphosphate (UTP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) caused transient opening of channels with linear conductances of approximately 18 and approximately 28 pS. Channels were permeable to Na+, K+, and Ca2+ and carried inward (depolarizing) current at the resting potential. Exposure to thapsigargin to increase cytosolic Ca2+ concentration ([Ca2+]i) opened similar channels, suggesting that opening is stimulated by a rise in [Ca2+]i. In subconfluent monolayers, ATP increased [Ca2+]i with half-maximal effects at approximately 7.4 microM; at 10(-4) M, the peak increase in [Ca2+]i was ATP > UTP > ATP gamma S >> 2-methylthioadenosine 5'-triphosphate, alpha,beta-methyleneadenosine 5'-triphosphate, and adenosine. The relative potency suggests that the effects are mediated by 5'-nucleotide receptors. In excised inside-out patches, channels were not activated by myo-inositol 1,4,5-trisphosphate (50-100 microM) or myo-inositol 1,3,4,5-trisphosphate (20 microM) but opened after increases in Ca2+ to greater than approximately 250 nM, consistent with a direct role for Ca2+ in channel opening. In intact cells, channel opening was followed by a prolonged refractory period. Protein kinase C appears to contribute by inhibition of the ATP-stimulated [Ca2+]i response and by direct inhibitory effects on the channel. These findings indicate that extracellular ATP leads to modulation of liver cell cation channels through activation of 5'-nucleotide receptors and are consistent with a model in which transient opening of channels is stimulated by a rise in [Ca2+]i and subsequent closure is mediated by protein kinase C-dependent pathways.

Stimulation of Ca2+ current by phorbol esters in rat myometrial cells is dependent on intracellular Ca2+ concentration

1996 ◽  
Vol 8 (8) ◽  
pp. 1147 ◽  
Author(s):  
M Kusaka ◽  
N Sperelakis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Voltage Clamp ◽  
Phorbol Esters ◽  
Cell Voltage ◽  
Muscle Cells ◽  
Whole Cell ◽  
Perforated Patch ◽  
Kinase C ◽  
Whole Cell Voltage Clamp

The effects of phorbol esters on the L-type Ca2+ current (ICa(L)) were investigated using nystatin-perforated patch and standard whole-cell voltage clamp in uterine smooth muscle cells isolated from late-pregnant rats. Using nystatin-perforated patch to maintain the integrity of the cytosol components, phorbol 12-myristate 13-acetate (PMA, 300 nM) increased ICa(L). When the standard whole-cell voltage clamp was used, the effect of PMA was dependent on the Ca2+ concentration in the pipette solution: PMA enhanced ICa(L) at pCa 6 and pCa 7 but not at pCa 10 or pCa 8. The effect of PMA was reversed by a selective inhibitor of protein kinase C, calphostin-C (500 nM). It is concluded that phorbol esters stimulate ICa(L) in uterine muscle cells and that the isoform of protein kinase C involved in this effect is Ca2+ dependent. This mechanism may be involved in the regulation of uterine contraction during pregnancy.

Protein kinase C stimulates Ca2+ current in pregnant rat myometrial cells

1994 ◽  
Vol 72 (11) ◽  
pp. 1304-1307 ◽  
Author(s):  
Keiichi Shimamura ◽  
Masumi Kusaka ◽  
Nicholas Sperelakis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Myometrial Cells ◽  
Kinase C ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent

The factors that regulate the voltage-dependent Ca2+ channels in pregnant uterine smooth muscle cells have not been elucidated, including any roles for protein kinase C (PKC). Therefore, the role of PKC in the regulation of the slow (L type) Ca2+ channels was examined in myometrial cells isolated from late pregnant (18–19 day) rat uterus, using the nystatin-perforated whole-cell voltage clamp. A PKC activator, phorbol 12, 13-dibutyrate (PDB), increased the L-type Ca2+ current (ICa(L)). Bath application of PDB (0.03 and 0.3 μM) increased the peak amplitude of ICa(L) by 21 ± 14% (n = 6) and 37 ± 8% (n = 9, p < 0.01), respectively. PDB did not change the holding current or shift the current–voltage relationship for ICa(L). The PKC inhibitors, H-7 (20 μM) or staurosporine (10 nM), reversed the effect of PDB. These results indicate that PKC may play a role in regulating Ca2+ channel function in pregnant rat myometrial cells and, therefore, may be involved in control of uterine contraction.Key words: protein kinase C, phorbol ester, calcium current, myometrial cell, nystatin-perforated patch, whole-cell voltage clamp.

Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA

1994 ◽  
Vol 24 (1-4) ◽  
pp. 295-300 ◽  
Author(s):  
Mutsumi Uchiyama ◽  
Keiko Hirai ◽  
Fumio Hishinuma ◽  
Hiroyuki Akagi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Xenopus Oocytes ◽  
Glycine Receptor ◽  
Down Regulation ◽  
Kinase C

Alpha 2 adrenoceptor potentiates glycine receptor-mediated taurine response through protein kinase A in rat substantia nigra neurons

1996 ◽  
Vol 76 (4) ◽  
pp. 2447-2454 ◽  
Author(s):  
J. Nabekura ◽  
T. Omura ◽  
N. Akaike
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Substantia Nigra ◽  
Glycine Receptor ◽  
Peak Amplitude ◽  
Facilitatory Effect ◽  
Equilibrium Potential ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Simultaneous Application

1. The modulatory effect of alpha 2 adrenoceptor on the taurine response was investigated in substantia nigra (SN) neurons acutely dissociated from the rat using a nystatin perforated-patch recording mode under voltage-clamp conditions. 2. Complete cross-desensitization was observed between 10(-3) M glycine and 3 x 10(-3) M taurine-induced currents. Both currents were antagonized by 10(-6) M strychnine, thus indicating that taurine acts on strychnine-sensitive glycine receptor on the SN neurons. 3. The simultaneous application of norepinephrine (NE) with prazosin (10(-5) M) and propranolol (10(-5) M) potentiated the taurine response (Itau) in an NE concentration-dependent manner at a holding potential (VH) of -40 mV. Clonidine mimicked the NE effect on the Itau, thus indicating the involvement of alpha 2 adrenoceptor activation in the potentiation of Itau. 4. Alpha 2 adrenoceptor activation by NE with prazosin and propranolol significantly potentiated the peak amplitude of Itau without shifting the taurine concentration-response relationships either to left or right side. The respective concentrations of taurine for the threshold, half maximal and maximal responses in the presence of 10(-4) M NE with prazosin (10(-5) M) and propranolol (10(-5) M) were 3 x 10(-5) M, 3.1 x 10(-4) M, and 3 x 10(-3) M. The same concentrations in the absence of NE were 3 x 10(-5) M, 3.2 x 10(-4) M, and 3 x 10(-3) M, respectively. 5. The reversal potentials of Itau with and without NE were very close to the theoretical Cl- equilibrium potential, thus indicating that the potentiation of Itau by alpha 2 adrenoceptor activation was due to an increase in the taurine-induced Cl- currents. 6. Forskolin (3 x 10(-5) M) and isobutylmethylxanthine (3 x 10(-5) M) suppressed the peak amplitude of Itau. In the presence of dibutyryl cyclic AMP (10(-4) M), which also suppressed Itau, alpha 2 adrenoceptor activation failed to potentiate Itau. 7. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89) mimicked the effect of alpha 2 adrenoceptor activation on Itau. In addition, the potentiation of Itau by alpha 2 adrenoceptor was not observed in the presence of 10(-6) M H-89. 8. The treatment of SN neurons with pertussis toxin (500 ng/ ml) for 18 h completely abolished the facilitatory effect of alpha 2 adrenoceptor on Itau. 9. These results suggest that the activation of alpha 2 adrenoceptor coupled with IAP-sensitive GTP binding protein decreases the intracellular cyclic AMP and cyclic AMP-dependent protein kinase activity, thus resulting in the potentiation of glycine receptor-mediated taurine response in rat SN neurons.

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