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At least 163 records · Page 9Linked to original sources
PubMed1992

Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes.

1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

CCK modulates inhibitory synaptic transmission in the solitary complex through CCKB sites.

The CCKB antagonists L-365, 260 and PD134308 and the CCKA antagonist L-364, 718 were applied to neurones of the rat solitary complex (SC) isolated in brainstem slices, while recording either intracellularly or by whole-cell patch-clamp. The CCKB antagonists increased the amplitude of spontaneous or solitary tract-evoked Cl(-)-dependent inhibitory synaptic events by 25 +/- 5% in 5/7 neurones. These events were identified as (1) reversed spontaneous potentials, (2) reversed multisynaptic potentials and (3) outward currents blocked by the GABAA antagonist bicuculline. The CCKB antagonists had no postsynaptic action and increased excitatory synaptic events by 16 +/- 5% in only 3/9 neurones. The CCKA antagonist depolarized neurones but had no effect on synaptic events. Results suggest that CCK, released from the SC tissue, modulates GABAergic interneurones through CCKB sites. This mechanism could contribute to panic attacks, probably mediated by CCKB receptors.

Animals↗
PubMed1992

Adenosine-dependent enhancement by methylxanthines of excitatory synaptic transmission in hippocampus of rats.

Whole-cell patch-clamp was used to investigate synaptic transmission in hippocampal slices. Excitatory post-synaptic currents (EPSCs) were facilitated by low (less than or equal to 1 microM) adenosine (Ado) concentrations, while high concentrations had well-known inhibitory effects on the EPSC. When added on the background of preapplied Ado, methylxanthines caused a large potentiation of EPSCs. At saturation, the enhanced EPSC could exceed the control almost by an order of magnitude. Pertussis toxin strongly impaired the ability of Ado to block EPSCs but did not augment the facilitatory effect. The two components of the EPSC mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors were facilitated simultaneously and in equal proportions.

Adenosine↗
PubMed1992

Characterization of the effects of a new Ca2+ channel activator, FPL 64176, in GH3 cells.

We examined the effects of the benzolpyrrole-type Ca2+ channel activator FPL 64176 on voltage-dependent L-type Ca2+ channels in rat anterior pituitary (GH3) cells. FPL 64176 increased K(+)-dependent Ca2+ influx into GH3 cells with an EC50 value of 1.2 x 10(-7) M but had no effect on the binding of [3H]PN200-110 to GH3 cell membranes at concentrations up to 10(-6) M. Whole-cell patch-clamp electrophysiology revealed that FPL 64176 (1 microM) increased L-type Ca2+ channel current amplitude and shifted the current-voltage relationship in the hyperpolarizing direction. Furthermore, Ca2+ channel current activation and deactivation were prolonged. Single-channel analysis showed that FPL 64176 increased both the probability of channel opening and the mean channel open time. Interestingly, the effect of FPL 64176 on channel open time was highly voltage dependent, with much longer openings being observed at more hyperpolarized potentials. We conclude that FPL 64176 represents a new class of L-type Ca2+ channel activator with a novel site and mechanism of action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.

Animals↗
PubMed1992

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.

Animals↗
PubMed1992

Modulation of sodium channel activity in frog ventricular cells by guanidyl-side armed cyclam.

The mechanism underlying the Na channel blocking action of guanidyl-side armed cyclam (G-cyclam) was studied using conventional patch-clamp methods. G-cyclam applied to the cytoplasmic surface of the membrane reduced the amplitude of single Na channel currents without inducing a flickering block. This effect was enhanced by depolarization and was fully reversible upon washout of the drug. The relationship between the concentration of G-cyclam and the reduction of unitary current could be expressed mathematically assuming one-to-one stoichiometry. During maximal suppression of the single channel current by G-cyclam approximately 40% of the current remained. Low concentration of G-cyclam (3 x 10(-4) M) prolonged, while higher concentration (3 x 10(-3) M) shortened the mean open time suggesting the involvement of two processes in the Na channel blocking action of this agent. It appears that low concentration of G-cyclam induce rapid and frequent transition between open and less conductive state resulting in a reduced current, and higher concentration make the channel nonconductive with slower single channel kinetics.

Animals↗
PubMed1992

Properties of the cGMP-activated channel of retinal on-bipolar cells.

Whole-cell patch-clamp recordings were obtained from on-bipolar cells in, or isolated from, retinal slices prepared from dogfish retina. The properties of the cGMP-activated conductance of on-bipolar cells were compared with that of rod photoreceptors. The on-bipolar cell cGMP-activated channel was blocked by L-cis-diltiazem, a block which was strongly voltage dependent. However, this channel is not identical with that of photoreceptors. The location of the L-cis-diltiazem blocking site and its accessibility in the channel are not the same as in rods. The voltage dependence of block suggests that the blocking site, although near the intracellular side of the channel, is accessible to the positively charged form of L-cis-diltiazem only from the outward facing side of the channel. Furthermore, in contrast to rod channels, the conductance of the on-bipolar cell channels is unaltered by the removal of external divalent cations.

Aminobutyrates↗
PubMed1992

Alpha 1-adrenoceptor stimulation enhances the delayed rectifier K+ current of guinea pig ventricular cells through the activation of protein kinase C.

The effect of alpha 1-adrenoceptor stimulation on the delayed rectifier K+ current (IK) was examined in isolated guinea pig ventricular cells by use of the patch-clamp method. IK was evoked by a 3-second depolarizing pulse from a holding potential of -30 mV in a Na(+)- and K(+)-free solution containing 3 microM nifedipine. Phenylephrine (30 microM) in the presence of propranolol (1 microM) produced an increase in IK. In five cells, phenylephrine increased the tail current of IK by 23 +/- 5%. This effect of phenylephrine was blocked by prazosin (0.3 microM), a selective alpha 1-blocker. Phenylephrine produced only a small effect on the voltage and time dependence of IK. Pretreatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10 microM) abolished the phenylephrine-induced increase in IK. In addition, pretreatment with a maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate (100 nM) abolished the phenylephrine-induced increase in IK. In conclusion, alpha 1-adrenoceptor stimulation increases IK in guinea pig cardiomyocytes. This alpha 1-adrenoceptor-mediated response may be related to an activation of protein kinase C. The increase in IK may explain a shortening of action potential duration observed after alpha 1-adrenoceptor stimulation in guinea pig cells.

Action Potentials↗
PubMed1992

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.

Animals↗
PubMed1992

Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava.

1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

Animals↗
PubMed1992

Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells.

We studied the developmental changes in the beta-adrenergic modulation of L-type calcium current (ICa) in enzymatically isolated adult (AD) and newborn (NB, 1-4-day-old) rabbit ventricular cells using the whole-cell patch-clamp method. ICa was measured as the peak inward current at a test potential of +15 mV by applying a 180-450-msec pulse from a holding potential of -40 mV with Cs(+)-rich pipettes and a K(+)-free bath solution at room temperature. In control, ICa density (obtained by normalizing ICa to the cell capacitance) was significantly higher in AD cells (5.5 +/- 0.2 [mean +/- SEM] pA/pF, n = 65) than in NB cells (2.6 +/- 0.1 pA/pF, n = 60). Isoproterenol (ISO, 1 nM-30 microM) increased ICa in a dose-dependent manner for both groups. The maximal effect (Emax) of ISO, expressed as percent increase in ICa over control levels, and the concentration for one half of the maximal effect (EC50) were 203% and 51 nM, respectively, for AD cells and 111% and 81 nM, respectively, for NB cells. The effect of ISO (1 microM) on ICa was decreased as the test potential was increased from -10 to +40 mV. However, the ratio of the percent increase in ICa for AD versus NB cells was almost constant (2.09-2.45) at each test potential. Dose-response curves of forskolin (FOR, 0.3-50 microM) gave Emax and EC50 of 268% and 0.74 microM, respectively, for AD cells and 380% and 1.15 microM, respectively, for NB cells. After stimulating ICa by 10 microM ISO, the addition of 10 microM FOR produced a further increase in ICa of only 12 +/- 2% in AD cells (n = 4) but a further increase of 140 +/- 41% in NB cells (n = 6). FOR (10 microM) did not produce any increase in ICa for AD and NB cells after stimulating ICa by intracellular application of 200 microM cAMP. ICa density stimulated by 10 microM ISO (17.8 +/- 1.1 pA/pF, n = 7), 10 microM FOR (21.0 +/- 1.3 pA/pF, n = 8), or 200 microM cAMP (18.0 +/- 1.3 pA/pF, n = 5) was equivalent in AD cells, whereas ICa density stimulated by 10 microM ISO (5.8 +/- 0.6 pA/pF, n = 9) was significantly lower than that stimulated by either 10 microM FOR (13.8 +/- 1.5 pA/pF, n = 7) or 200 microM cAMP (13.4 +/- 0.7 pA/pF, n = 7) in NB cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1992

The glutamate-receptor linked cGMP cascade of retinal on-bipolar cells is pertussis and cholera toxin-sensitive.

Whole-cell patch-clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of the A-subunit of pertussis toxin in the patch-pipette solution resulted in an increase in inward current and membrane conductance, and a block of light-evoked currents of on-bipolar cells. The opposite effect was obtained with the A-subunit of cholera toxin, which blocked light responses, and induced an outward current and a decrease in membrane conductance. These actions were NAD+ dependent. The results show that the G-protein(s) linking glutamate receptors to a cGMP cascade in on-bipolar cells possess sites which are ADP-ribosylated by pertussis and cholera toxins, with no homology to the adenylate cyclase system but possibly with a homology to transducin. Furthermore, inclusion of H-7, a kinase inhibitor in the patch-pipette solution, or of a non-hydrolysable ATP analogue (AMP-PNP) had no effect on light responses, membrane conductance or dark current of on-bipolar cells, suggesting that the components of this cGMP cascade are unlikely to be regulated by protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.

Action Potentials↗
PubMed1992

Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea.

Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-clamp, by recording onset latencies and response amplitudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-clamp at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.

Acetylcholine↗
PubMed1992

ATP mediates excitatory synaptic transmission in mammalian neurones.

Adenosine 5'-triphosphate (ATP, 0.1-100 microM), produced inward currents in patch-clamped coeliac neurones from guinea-pig when studied in either the whole cell configuration or in excised (outside-out) patches. The P2-purinoceptor antagonists suramin (80-230 microM) or reactive blue 2 (2-20 microM) depressed the ATP-induced currents but not those produced by acetylcholine. Excitatory post-synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current-voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.

Acetylcholine↗
PubMed1992

Permeability and Mg2+ blockade of histamine-operated cation channel in endothelial cells of rat intrapulmonary artery.

1. In the cell-attached and inside-out patch-clamp experiments using undispersed endothelial cells of the rat intrapulmonary artery, the majority of channels were cation selective. 2. Under physiological ionic conditions, the I-V relationship for the inward currents fell to -80 mV and the slope conductance was 22.5 pS. There was an inward rectification and the outward currents were smaller than the inward currents. 3. Under symmetric high-K+ conditions, the slope conductance for the inward currents was 26.4 pS and the inward rectification was observed when the high-K+ solution contained 1 mM-Mg2+. The channel activity was weakly voltage dependent at negative membrane potentials, while it was much enhanced at positive potentials. 4. The channel activity did not depend on intracellular Ca2+ concentrations. 5. Mg2+ was not only impermeant, it also blocked this channel in a voltage-dependent manner and rectifications appeared in the I-V relationship. Mg2+ blocked the channel from both sides of the membrane. 6. Ca2+ permeated this channel and the permeability ratios calculated from the reversal potentials using the constant-field theory were; PK:PNa:PCa = 1:1:15.7. 7. Histamine but not acetylcholine applied to the pipette activated this channel. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) applied to the intracellular surface of the patch did not mimic the effect of histamine. 8. Thus, in the endothelial cell membrane of the rat intrapulmonary artery, there exists a cation channel which is selective to Ca2+ but also permeable to Na+ and K+. This channel has inward rectifying properties, possibly due to intracellular Mg2+. Histamine, but not acetylcholine, activates this cation channel to elevate endothelial [Ca2+]i.

Acetylcholine↗
PubMed1992

Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells.

Using whole cell patch-clamp and perforated patch recording techniques on human cystic fibrosis (CF) and non-CF airway epithelial cells, we sought to determine whether a single Cl- conductance (GCl) could be modulated via different regulatory pathways or whether multiple conductances could be identified. Cl- current in both CF and non-CF cells was activated by cellular swelling as well as by an elevation in intracellular calcium ([Ca2+]i). While the adenosine 3',5'-cyclic monophosphate (cAMP)-activated GCl was absent in CF cells, its activation in non-CF cells was only observed in the perforated patch configuration at lower temperatures (24 degrees C) or infrequently in the whole cell configuration at elevated temperatures (33 degrees C). Currents activated by all three regulatory pathways were sensitive to the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Further increases in current activation could be produced by cellular swelling after maximal Ca2+ or cAMP-induced current activation. Intracellular application of a peptide inhibitor of Ca(2+)-calmodulin-dependent protein kinase selectively blocked the Ca(2+)-dependent current activation while leaving the swelling-induced current increase intact. These results are consistent with the presence of multiple anion conductances in both CF and non-CF airway cells. The heterogeneity of the responses to the three regulatory stimuli, however, prevented the correlation of a specific anion conductance with a separate modulatory pathway based on characteristic voltage-dependent kinetics and conductance.

Calcium↗