Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner

2014 ◽  
Vol 306 (9) ◽  
pp. C856-C863 ◽  
Author(s):  
Rui Feng ◽  
Jianjun Xu ◽  
Etsuko Minobe ◽  
Asako Kameyama ◽  
Lei Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Guinea Pig ◽  
Terminal Region ◽  
Atp Binding ◽  
Dependent Manner ◽  
Channel Activity ◽  
Fusion Peptides ◽  
Patch Clamp Technique ◽  
Binding Effect ◽  
Dose Dependent

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca2+ channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca2+ channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca2+ was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6–140) and proximal COOH-terminal region (amino acids 1,509–1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca2+ dependent.

scholarly journals Metabolic inhibition impairs ATP-sensitive K+ channel block by sulfonylurea in pancreatic β-cells

1998 ◽  
Vol 274 (1) ◽  
pp. E38-E44 ◽  
Author(s):  
Eri Mukai ◽  
Hitoshi Ishida ◽  
Seika Kato ◽  
Yoshiyuki Tsuura ◽  
Shimpei Fujimoto ◽  
...  
Keyword(s):  
Metabolic Inhibition ◽  
K Channel ◽  
High Dose ◽  
Dependent Manner ◽  
Channel Activity ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Dose Dependent

The effect of metabolic inhibition on the blocking of β-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 μM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 μM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATPchannel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic β-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

scholarly journals Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

2012 ◽  
Vol 302 (9) ◽  
pp. C1361-C1370 ◽  
Author(s):  
Wenkuan Xin ◽  
Qiuping Cheng ◽  
Rupal P. Soder ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Detrusor Smooth Muscle ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Pde Inhibition

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca2+-activated K+ (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca2+ sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

Polydatin Attenuates Carbachol-induced Contraction of Guinea Pig Gallbladder

2019 ◽  
Vol 18 (1) ◽  
pp. 34-38
Author(s):  
Chen Lei ◽  
Pan Xiang ◽  
Shen Yonggang ◽  
Song Kai ◽  
Zhong Xingguo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Smooth Muscles ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Dependent Manner ◽  
Underlying Mechanisms ◽  
Dose Dependent

The aim of this study was to determine whether polydatin, a glucoside of resveratrol isolated from the root of Polygonum cuspidatum, warranted development as a potential therapeutic for ameliorating the pain originating from gallbladder spasm disorders and the underlying mechanisms. Guinea pig gallbladder smooth muscles were treated with polydatin and specific inhibitors to explore the mechanisms underpinning polydatin-induced relaxation of carbachol-precontracted guinea pig gallbladder. Our results shown that polydatin relaxed carbachol-induced contraction in a dose-dependent manner through the nitric oxide/cyclic guanosine monophosphate/protein kinase G and the cyclic adenosine monophosphate/protein kinase A signaling pathways as well as the myosin light chain kinase and potassium channels. Our findings suggested that there was value in further exploring the potential therapeutic use of polydatin in gallbladder spasm disorders.

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 Simonkolleite Coating on Poly(Amino Acids) to Improve Osteogenesis and Suppress Osteoclast Formation in Vitro

Polymers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1505 ◽  
Author(s):  
Shuyang Li ◽  
Xingtao Chen ◽  
Xiaomei Wang ◽  
Yi Xiong ◽  
Yonggang Yan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Osteoclast Formation ◽  
Mouse Bone Marrow ◽  
Graft Material ◽  
Dependent Manner ◽  
E Coli ◽  
Ft Ir ◽  
Dose Dependent ◽  
Relative Cell Viability

Zinc can enhance osteoblastic bone formation and stimulate osteogenic differentiation, suppress the differentiation of osteoclast precursor cells into osteoclasts, and inhibit pathogenic bacterial growth in a dose-dependent manner. In this study, simonkolleite, as a novel zinc resource, was coated on poly (amino acids) (PAA) via suspending PAA powder in different concentrations of zinc chloride (ZnCl2) solution, and the simonkolleite-coated PAA (Zn–PAA) was characterized by SEM, XRD, FT-IR and XPS. Zinc ions were continuously released from the coating, and the release behavior was dependent on both the concentration of the ZnCl2 immersing solution and the type of soak solutions (SBF, PBS and DMEM). The Zn–PAA was cultured with mouse bone marrow stem cells (BMSCs) through TranswellTM plates, and the results indicated that the relative cell viability, alkaline phosphatase (ALP) activity and mineralization of BMSCs were significantly higher with Zn–PAA as compared to PAA. Moreover, the Zn–PAA was cultured with RAW264.7 cells, and the results suggested an inhibiting effect of Zn–PAA on the cell differentiation into osteoclasts. In addition, Zn–PAA exhibited an antibacterial activity against both S. aureus and E. coli. These findings suggest that simonkolleite coating with certain contents could promote osteogenesis, suppress osteoclast formation and inhibit bacteria, indicating a novel way of enhancing the functionality of synthetic bone graft material and identifying the underline principles for designing zinc-containing bone grafts.

Delayed-rectifier potassium channel activity in isolated membrane patches of guinea pig ventricular myocytes

1991 ◽  
Vol 260 (4) ◽  
pp. H1390-H1393 ◽  
Author(s):  
K. B. Walsh ◽  
J. P. Arena ◽  
W. M. Kwok ◽  
L. Freeman ◽  
R. S. Kass
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Time Dependent ◽  
Delayed Rectifier ◽  
Potential Range ◽  
Ammonium Compound ◽  
Channel Activity ◽  
Patch Clamp Technique

When the patch-clamp technique was used, a slowly activating, time-dependent outward current was identified in both cell-attached and excised membrane patches obtained from guinea pig ventricular myocytes. This macroscopic patch current was present in approximately 50% of patches studied and could be observed both in the presence and absence of unitary single channel activity (i.e., ATP-sensitive K+ channels). The time course of activation of the patch current resembled that of the whole cell delayed-rectifier K+ current (IK) recorded under similar ionic conditions, and the patch current and IK were activated over a similar membrane potential range. The time-dependent patch current could be eliminated when the Nernst potential for K+ equaled that of the pulse voltage. The patch current was inhibited by external addition of the tertiary ammonium compound LY 97241 (50 microM) and was augmented after internal application of the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (500 nM). Deactivating tail currents with kinetics similar to those of IK could be recorded to cell-attached and excised patches. Unitary single channel events underlying the time-dependent patch current could not be resolved despite various attempts to increase single channel conductance. Thus our results suggest that a major component of delayed rectification in guinea pig ventricular cells is due to the activity of a high-density, extremely low conductance K+ channel.

Endothelium-dependent and -independent relaxations to adenosine in guinea pig aorta

1990 ◽  
Vol 259 (1) ◽  
pp. H62-H67 ◽  
Author(s):  
J. P. Headrick ◽  
R. M. Berne
Keyword(s):  
Guinea Pig ◽  
Sodium Nitroprusside ◽  
Maximal Response ◽  
Dependent Manner ◽  
Prostaglandin F2 Alpha ◽  
Endothelial Response ◽  
Alpha Response ◽  
Dose Dependent ◽  
Subtype 3 ◽  
Indomethacin Treatment

Effects of endothelial removal and hypoxia on responses to adenosine, 5'-(N-ethylcarboxamido)-adenosine (NECA), 2-chloroadenosine, N6-cyclohexyladenosine (CHA), sodium nitroprusside, and acetylcholine were examined in guinea pig aortic rings. Rings contracted with 2 microM prostaglandin F2 alpha (PGF2 alpha) relaxed in a dose-dependent manner in response to all drugs. The order of potency of adenosine compounds was NECA greater than 2-chloroadenosine greater than adenosine greater than CHA. Endothelial rubbing potentiated the PGF2 alpha response by 11 +/- 3%, eliminated the acetylcholine (ACh) response, but had no effect on nitroprusside and CHA responses. Responses to adenosine, NECA, and 2-chloroadenosine were significantly depressed by rubbing (P less than 0.05). Oxyhemoglobin (5 microM) and metyrapone (0.1 mM) reduced ACh responses in intact rings but had no effect on the adenosine and nitroprusside responses. Indomethacin treatment (10 microM) did not alter ACh, nitroprusside, or adenosine responses in intact rings. Hypoxia (10% O2) potentiated maximal responses to adenosine (+26 +/- 3%) and nitroprusside (+28 +/- 4%) in intact and rubbed rings and reduced the maximal response to ACh in intact rings (-28 +/- 3%). It is concluded that 1) adenosine mediates relaxation in guinea pig aorta by endothelial-dependent and -independent mechanisms, 2) receptors involved in both endothelial-dependent and -independent relaxations are characteristic of the A2 adenosine subtype, 3) the endothelial response appears unrelated to EDRF or prostanoid release, and 4) the adenosine response is significantly potentiated by hypoxia.

Activation of Phosphatidylinositol-Linked Novel D1 Dopamine Receptors Inhibits High-Voltage-Activated Ca2+ Currents in Primary Cultured Striatal Neurons

2009 ◽  
Vol 101 (5) ◽  
pp. 2230-2238 ◽  
Author(s):  
Li-Qun Ma ◽  
Chao Liu ◽  
Fang Wang ◽  
Na Xie ◽  
Jun Gu ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
High Voltage ◽  
Dependent Manner ◽  
Striatal Neurons ◽  
Inhibitory Effects ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
Dose Dependent

Recent evidences indicate the existence of a putative novel phosphatidylinositol (PI)-linked D1 dopamine receptor that mediates excellent anti-Parkinsonian but less severe dyskinesia action. To further understand the basic physiological function of this receptor in brain, the effects of a PI-linked D1 dopamine receptor-selective agonist 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) on high-voltage activated (HVA) Ca2+ currents in primary cultured striatal neurons were investigated by whole cell patch-clamp technique. The results indicated that stimulation by SKF83959 induced an inhibition of HVA Ca2+ currents in a dose-dependent manner in substance-P (SP)-immunoreactive striatal neurons. Application of D1 receptor, but not D2, α1 adrenergic, 5-HT receptor, or cholinoceptor antagonist prevented SKF83959-induced reduction, indicating that a D1 receptor-mediated event assumed via PI-linked D1 receptor. SKF83959-induced inhibitory modulation was mediated by activation of phospholipase C (PLC), mobilization of intracellular Ca2+ stores and activation of calcineurin. Furthermore, the inhibitory effects were attenuated significantly by the L-type calcium channel antagonist nifedipine, suggesting that L-type calcium channels involved in the regulation induced by SKF83959. These findings may help to further understand the functional role of the PI-linked dopamine receptor in brain.

Depression of delayed outward K+ current by Co2+ in guinea pig ventricular myocytes

1991 ◽  
Vol 261 (1) ◽  
pp. C23-C31 ◽  
Author(s):  
Z. Fan ◽  
M. Hiraoka
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Hill Coefficient ◽  
Maximal Response ◽  
Fluctuation Analysis ◽  
Dependent Manner ◽  
Surface Charges ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
K Current

Effects of Co2+ on the delayed outward K+ current (IK) in guinea pig ventricular myocytes were studied using the whole cell patch-clamp technique. IK was activated by depolarizing voltage pulses positive to -30 mV and reached half-maximal activation at +24 mV. Co2+ shifted the activation curve to a more depolarized voltage range in a concentration-dependent manner, with a Co2+ concentration at which half-maximal response occurs (IC50) of 8 mM and a saturation value of +38 mV. The voltage dependency of IK gatings showed a shift similar to that of activation. In both cases the shift could be explained by screening of surface potential. The density of total negative surface charges sensed by Co2+ was estimated to be 1 e/225 A2. Co2+ also reduced the fully activated IK [IK(full)], and the dose-response curve had a Hill coefficient of 0.5 and an IC50 of 1 mM at 0 mV. Depression of IK(full) was mainly voltage independent. The single-channel unitary current estimated by fluctuation analysis was approximately 0.1 pA at -30 mV either in the absence or presence of Co2+. Therefore, the depression of IK(full) is due to an equivalent reduction in the number of functional channels. It is concluded that Co2+ depressed IK through multiple mechanisms.

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