Expression of 5-HT3 receptors in PC12 cells treated with NGF and 8-Br-cAMP

1992 ◽  
Vol 67 (4) ◽  
pp. 812-819 ◽  
Author(s):  
K. Furukawa ◽  
N. Akaike ◽  
H. Onodera ◽  
K. Kogure
Keyword(s):  
Pc12 Cells ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Inactivation Kinetics ◽  
Induced Response ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inward Current

1. To determine the functional development of neurons, we applied nerve growth factor (NGF) or 8-bromo-cyclic-adenosine monophosphate (8-Br-cAMP) to PC12 cells and recorded the 5-hydroxytryptamine (5-HT)-induced response by the use of a patch-clamp technique. 2. Cultured PC12 cells expressed 5-HT-sensitive receptors, which are almost absent in untreated cells, in the continuous presence of NGF or 8-Br-cAMP for a period of 10 days. 3. Activation of the receptors by 5-HT produced a transient inward current. In a K(+)-free solution, the reversal potential (E5-HT) of I5-HT was +10.3 mV, and the current-voltage (I-V) relation showed inward rectification at positive potentials. 4. The permeability ratio for monovalent cations was Na+:Li+:K+:Rb+:Cs+ = 1:1.19:0.89:0.94:0.91, indicating that a 5-HT-induced current is passing through the ligand-gated large cation channel. 5. 2-Methyl-5-HT, a specific 5-HT3 agonist, induced a similar inward current, even though the current amplitude was smaller and the activation and inactivation kinetics were slower than those of 5-HT. 6. ICS-205-930, a specific 5-HT3 antagonist, inhibited the 5-HT-induced current in a concentration-dependent manner with a noncompetitive inhibition profile. Spiperone, a 5-HT1 and 5-HT2 families antagonist, and ketanserine, 5-HT2 family antagonist, did not affect the 5-HT-induced response. 7. The time to peak (tp) as well as fast and slow time constants (tau if and tau is) decreased with increasing 5-HT concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucose deprivation on NMDA-induced current and intracellular Ca2+ in rat substantia nigra neurons

1996 ◽  
Vol 75 (2) ◽  
pp. 740-749 ◽  
Author(s):  
Y. Nakashima ◽  
H. Ishibashi ◽  
N. Harata ◽  
N. Akaike
Keyword(s):  
Substantia Nigra ◽  
Outward Current ◽  
Glucose Deprivation ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inward Current ◽  
Rat Substantia Nigra

1. The effects of glucose deprivation on N-methyl-D-asparate (NMDA)-induced current (INMDA) and the intracellular free Ca2+ concentration ([Ca2+]i) in the acutely dissociated rat substantia nigra neurons were investigated using the nystatin-perforated patch-clamp technique under voltage clamp and the microfluometry with a fluorescent probe, Indo-1. 2. Application of NMDA induced a peak and a successive steady-state inward current, and an outward current immediately after washout at a holding potential of -40 mV. The amplitudes of the three current components of INMDA were increased by increasing the concentrations of NMDA with half-maximum concentrations (EC50s) of 1.1 x 10(-4) M, 1.2 x 10(-4) M, and 1.6 x 10(-4) M, respectively. 3. The reversal potentials of the peak inward and outward currents were -4 +/- 3 (SE) mV and -76 +/- 2 mV, respectively. The latter was close to the theoretical K+ equilibrium potential (-82 mV). 4. The outward current was potentiated by increase in extracellular Ca2+ concentration and was blocked by Cs+ internal solution and suppressed by 5 x 10(-3) M tetraethylammonium chloride and 10(-7) M charybdotoxin, indicating that it was Ca(2+)-activated K+ current. 5. Application of NMDA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 3.9 x 10(-5) M. 6. Depriving the external solution of glucose induced a slowly developing outward current and increased the basal level of [Ca2+]i. It also prolonged the NMDA-induced outward current without affecting the peak inward current, and prolonged the NMDA-induced increase in [Ca2+]i without changing the peak [Ca2+]i. 7. These findings suggest that the deprivation of glucose did not affect the NMDA-induced influx of Ca2+ into the cells, but it inhibited Ca2+ clearance by affecting the efflux of Ca2+ to the extracellular space, reuptake into the intracellular Ca2+ stores, and/or active extrusion from intracellular stores.

ATP-gated current in dissociated rat nucleus solitarii neurons

1992 ◽  
Vol 68 (3) ◽  
pp. 778-785 ◽  
Author(s):  
S. Ueno ◽  
N. Harata ◽  
K. Inoue ◽  
N. Akaike
Keyword(s):  
Purinergic Receptor ◽  
Reversal Potential ◽  
External Solution ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Inactivation Kinetics ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Continuous Presence

1. The excitatory response of extracellularly applied ATP was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole-cell configuration using the ”concentration-clamp” technique. 2. At a holding potential of -70 mV, 100 microM ATP evoked inward current that was slowly desensitized in the continuous presence of ATP. The ATP-gated current increased in a concentration-dependent manner over the concentration range between 10 microM and 1 mM. The half-maximum concentration was 31 microM and the Hill coefficient was 1.2. 3. The potency of ATP analogues for the purinergic receptor was in the order of ATP = 2-methylthio-ATP much greater than ADP greater than alpha,beta-methylene ATP. Neither adenosine nor AMP evoked any responses. The order was consistent with a P2y receptor subtype. 4. The current-voltage relationship for the 100 microM ATP response showed a clear inward rectification at positive potentials beyond -50 mV. The reversal potential of the ATP-gated current was +13 mV. 5. The time constants of activation and inactivation of the ATP-gated current solution were dependent on the extracellular ATP concentration, and both kinetics became faster at higher ATP concentrations. 6. The ATP-gated current was also elicited in an external solution containing Ca2+ as a permeable cation. The inactivation kinetics in an external solution containing 75 mM Ca2+ were faster than those in an external solution with 150 mM Na+. 7. Calculated relative permeability ratios were PNa/PCs = 1.64 ([Na+]o = 30-150 mM), PCa/PCs = 2.17 ([Ca2+]o = 2 mM). Anions were not measurably permeable in this preparation.

ATP-induced inward current in neurons freshly dissociated from the tuberomammillary nucleus

1994 ◽  
Vol 71 (3) ◽  
pp. 868-873 ◽  
Author(s):  
K. Furukawa ◽  
H. Ishibashi ◽  
N. Akaike
Keyword(s):  
Purinergic Receptor ◽  
Reversal Potential ◽  
External Solution ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Induced Current ◽  
Excitatory Effect ◽  
Inward Current ◽  
Tuberomammillary Nucleus

1. Neurons in the tuberomammillary nucleus (TMN), which are considered to be histaminergic, were dissociated and their response to extracellularly applied ATP was investigated in the nystatin-perforated patch recording mode under voltage-clamp condition. 2. ATP induced a sustained inward current that was slowly desensitized at a holding potential of -60 mV. 3. The ATP response increased in a concentration-dependent manner. The half-maximum concentration (EC50) was 44 microM and the Hill coefficient was 1.8. 4. The potency of ATP analogues was in the order of ATP > or = 2-methylthio-ATP >> alpha, beta-methylene ATP > or = ADP. Neither adenosine nor AMP induced any response. The results suggest that the purinergic receptor in TMN neurons is P2y. 5. The current-voltage relationship for the 100 microM ATP showed a significant inward rectification at a potential more positive than -20 mV in an external solution with 150 mM Na+, but a significant rectification current was not observed in an external solution with 150 mM Cs+. The change in the reversal potential of the ATP response (EATP) to a 10-fold change of extracellular Na+ concentration was 56 mV, indicating that the ATP-induced current is highly selective for Na+ over Cl-. 6. The permeability ratio for cations was Na+:Li+:K+:Rb+: Cs+:Ca2+ = 2.16:1.36:1.68:1.54:1:2.55, indicating that the ATP-induced current is passing through the ligand-gated nonselective cation channel. 7. These results suggest that ATP has an excitatory effect on the TMN neurons by opening nonselective cation channels.

gamma-Aminobutyric acid-induced response in rat dissociated paratracheal ganglion cells

1992 ◽  
Vol 67 (5) ◽  
pp. 1367-1374 ◽  
Author(s):  
S. Itabashi ◽  
K. Aibara ◽  
H. Sasaki ◽  
N. Akaike
Keyword(s):  
Response Curve ◽  
Ganglion Cells ◽  
Aminobutyric Acid ◽  
Equilibrium Potential ◽  
Induced Response ◽  
Dependent Manner ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Concentration Response Curve

1. The pharmacologic properties of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in the paratracheal ganglion cells freshly dissociated from 7- to 10-day-old rat trachea in a whole-cell recording mode by the use of a conventional patch-clamp technique. 2. GABA- and muscimol-induced currents increased sigmoidally in a concentration-dependent manner, and both currents reversed at approximately -3 mV, which was close to the Cl- equilibrium potential (ECl). 3. Strychnine (STR) at low concentration and bicuculline (BIC) inhibited GABA response competitively, whereas STR at the higher concentrations, benzylpenicillin (PCG), or picrotoxin (PTX) inhibited noncompetitively. Inhibition of GABA response by PCG but not other antagonists was voltage dependent, indicating that PCG acts as a Cl- channel blocker. 4. The concentration-response curve of pentobarbital sodium (PB)-induced ICl was bell shaped. At concentrations higher than 10(-3) M, both the peak and plateau currents decreased, and a transient "hump" current appeared immediately after washing out PB. In the presence of PB, the concentration-response curve of GABA shifted toward left without changing the maximum response. 5. Although diazepam (DZP) at concentration used did not induce a response, it potentiated the GABA response in a concentration-dependent manner between 10(-8) and 10(-6) M. DZP also caused a parallel shift toward left in the concentration-response curve of GABA. 6. PB or DZP further enhanced the GABA response in the presence of the other agent. 7. It is concluded that the properties of GABAA receptors in the paratracheal ganglion cells are essentially similar to those reported in other preparations.

ATP-induced current in isolated outer hair cells of guinea pig cochlea

1990 ◽  
Vol 63 (5) ◽  
pp. 1068-1074 ◽  
Author(s):  
T. Nakagawa ◽  
N. Akaike ◽  
T. Kimitsuki ◽  
S. Komune ◽  
T. Arima
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Reversal Potential ◽  
Outer Hair Cells ◽  
Hill Coefficient ◽  
Induced Response ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Positive Direction ◽  
Guinea Pig Cochlea

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor

2001 ◽  
Vol 281 (4) ◽  
pp. L931-L940 ◽  
Author(s):  
X. W. Fu ◽  
D. Wang ◽  
J. Pan ◽  
S. M. Farragher ◽  
V. Wong ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Whole Cell Patch Clamp ◽  
Cell Plasma ◽  
Neuroepithelial Body ◽  
Inward Currents ◽  
Type 3

Serotonin (5-HT) type 3 receptor (5-HT3-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT3-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT3-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT3-R) and confocal microscopy localized 5-HT3-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT3-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5–150 μM) and 5-HT3-R agonist 2-methyl-5-HT (5–150 μM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 ± 5.9%) by the specific 5-HT3-R antagonist ICS-205–930 (50 μM), whereas katanserin and p-4-iodo- N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}- N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca2+ and Mg2+ in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT3-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.

Dual effect of glycine on isolated rat suprachiasmatic neurons

1991 ◽  
Vol 260 (2) ◽  
pp. C213-C218 ◽  
Author(s):  
C. Ito ◽  
M. Wakamori ◽  
N. Akaike
Keyword(s):  
Neural Activity ◽  
Excitatory Amino Acids ◽  
Pharmacological Properties ◽  
Dependent Manner ◽  
Intracellular Recordings ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Dual Effect ◽  
Concentration Dependent Manner ◽  
Inhibitory Neurotransmitter

Pharmacological properties of strychnine-sensitive and -insensitive glycine receptors have been investigated in rat suprachiasmatic nucleus (SCN) neurons. Because the SCN neurons were too small for stable intracellular recordings by the glass-microelectrode technique, a conventional whole cell mode patch-clamp technique was employed on the acutely dissociated SCN neurons. Dissociated SCN neurons were morphologically heterogeneous and could be distinguished into several types. All cells responded to glycine in a concentration-dependent manner. The glycine-induced current was primarily Cl- sensitive and competitively blocked by strychnine. The SCN neurons also responded to excitatory amino acids: glutamate, quisqualate, kainate, and N-methyl-D-aspartate (NMDA). Responses to glutamate and aspartate, which are endogenous neurotransmitter candidates, were enhanced by adding glycine. Glycine especially augmented the maximum response to NMDA in a full concentration range. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) did not suppress the strychnine-sensitive glycine response but did suppress the strychnine-insensitive NMDA response in a competitive manner for glycine. The results suggest that glycine influences neural activity in the SCN as a classical inhibitory neurotransmitter and an excitatory neuromodulator.

Functional Expression of Three P2X2 Receptor Splice Variants From Guinea Pig Cochlea

2000 ◽  
Vol 83 (3) ◽  
pp. 1502-1509 ◽  
Author(s):  
Chu Chen ◽  
Margarett S. Parker ◽  
Anthony P. Barnes ◽  
Prescott Deininger ◽  
Richard P. Bobbin
Keyword(s):  
Guinea Pig ◽  
Splice Variants ◽  
Reversal Potential ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Human Embryonic Kidney Cells ◽  
Guinea Pig Cochlea

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X2 receptor splice variants, P2X2–1, P2X2–2, and P2X2–3, were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X2 receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage ( I-V) relationships for the ATP-gated current show that the three subtypes of the P2X2 receptor had a similar reversal potential and an inward rectification index ( I 50 mV/ I −50 mV). However, the ATP-induced currents in cells expressing P2X2–1 and P2X2–2 variants were large and desensitized rapidly whereas the current in those cells expressing the P2X2–3 variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > α,β -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X2splice variants from guinea pig cochlea, P2X2–1, P2X2–2, and P2X2–3, can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X2receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

P2X receptors in mouse Leydig cells

2006 ◽  
Vol 290 (4) ◽  
pp. C1009-C1017 ◽  
Author(s):  
Luiz Artur Poletto Chaves ◽  
Endrigo Piva Pontelli ◽  
Wamberto Antonio Varanda
Keyword(s):  
Leydig Cells ◽  
Pyridoxal Phosphate ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Apparent Dissociation Constant ◽  
Whole Cell

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 μM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5′- O-(3-thiotriphosphate) (ATPγS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response ( Kd) of 44, 110, and 637 μM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ≥ ATPγS > 2-MeS-ATP > 2′,3′- O-(4-benzoylbenzoyl)-ATP (BzATP). αβ-Methylene-ATP (αβ-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (p Ka) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability ( P)Ca/ PNa = 5.32], but not to chloride ( PCl/ PNa = 0.03) or N-methyl-d-glucamine (NMDG) ( PNMDG/ PNa = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between −90 and −50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (τ) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X2.

Crocin, a carotenoid component of Crocus cativus, exerts inhibitory effects on L-type Ca2+ current, Ca2+ transient, and contractility in rat ventricular myocytes

2016 ◽  
Vol 94 (3) ◽  
pp. 302-308 ◽  
Author(s):  
Tao Liu ◽  
Xi Chu ◽  
Hua Wang ◽  
Xuan Zhang ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Reversal Potential ◽  
Inhibitory Effect ◽  
Crocus Sativus ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes ◽  
Inotropic Effects ◽  
Cardioprotective Effects

Crocin, a carotenoid component of Crocus sativus L. belonging to the Iridaceae family, has demonstrated cardioprotective effects. To investigate the cellular mechanisms of these cardioprotective effects, here we studied the influence of crocin on L-type Ca2+current (ICa-L), intracellular Ca2+ ([Ca2+]i), and contraction of isolated rat cardiomyocytes by using the whole-cell patch-clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier systems. Crocin inhibited ICa-L in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) of 45 μmol/L and the maximal inhibitory effect of 72.195% ± 1.54%. Neither current–voltage relationship of ICa-L, reversal potential of ICa-L, nor the activation/inactivation of ICa-L was significantly changed. Crocin at 1 μmol/L reduced cell shortening by 44.64% ± 2.12% and the peak value of the Ca2+ transient by 23.66% ± 4.52%. Crocin significantly reduced amplitudes of myocyte shortening and [Ca2+]i with an increase in the time to reach 10% of the peak (Tp) and a decrease in the time to 10% of the baseline (Tr). Thus, the cardioprotective effects of crocin may be attributed to the attenuation of [Ca2+]i through the inhibition of ICa-L in rat cardiomyocytes and negative inotropic effects on myocardial contractility.

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