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At least 73 records · Page 4Linked to original sources
PubMed1992

Two types of high-threshold calcium currents inhibited by omega-conotoxin in nerve terminals of rat neurohypophysis.

1. The neurohypophysis comprises the nerve terminals of hypothalamic neurosecretory cells, which contain arginine vasopressin (AVP) and oxytocin. The secretory terminals of rat neurohypophyses were acutely dissociated. The macroscopic calcium currents (ICa) of these isolated peptidergic terminals were studied using 'whole-cell' patch-clamp recording techniques. 2. There are two types ('Nt' (where the subscript 't' denotes terminal) and 'L') of high-threshold voltage-activated ICa in the terminals, which can be distinguished by holding at different potentials i.e. -90 and -50 mV. Replacement of Ca2+ in the bathing solution by Ba2+ increased the amplitude of ICa, primarily due to an increase in the L-type component. Both inward currents were eliminated by adding 50 microM-Cd2+ or when in a Ca(2+)-free bathing solution. 3. omega-Conotoxin GVIA (omega-CgTx) has been widely used as a Ca2+ channel blocker. However, whether this toxin can discriminate between different types of Ca2+ channels is still a subject of controversy. We applied omega-CgTx over a wide range of concentrations (0.01-2 microM) to examine its effects on both Nt- and L-type ICa in these terminals. At a concentration of 30 nM, omega-CgTx selectively reduced, by 48%, the amplitude of Nt-type ICa. In contrast, a higher concentration (300 nM) of omega-CgTx was necessary to inhibit the L-type ICa. 4. omega-CgTx inhibited both Nt- and L-type ICa in a dose-dependent manner, and the half-maximum inhibition (IC50) of the ICa by the toxin was 50 and 513 nM, respectively, which was approximately a tenfold difference. The reduction in both types of currents did not result from any shift in their current-voltage or steady-state inactivation relationships. 5. In contrast, omega-CgTx, at a concentration of 300 nM, had no effect on the tetrodotoxin-sensitive sodium current (INa) of the isolated peptidergic nerve terminals. Furthermore, omega-CgTx did not reduce the long-lasting, non-inactivating ICa in the isolated non-neuronal secretory cells of the pars intermedia (PI) (intermediate lobe of the pituitary). 6. Our studies suggest that omega-CgTx might exert specific blocking effects on both Nt- and L-type Ca2+ channels, but that in the isolated peptidergic nerve terminals, the Nt-type component is more susceptible to this toxin.

Action Potentials↗
PubMed1992

Stretch-activated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries.

1. Single stretch-activated (SA) channels have been studied in isolated brain capillary endothelial cells as well as in the antiluminal membrane of intact porcine cerebral capillaries using the patch-clamp recording technique. 2. The SA channels were found to be cation selective and permeable to Na+, K+, Ba2+ and Ca2+. 3. With monovalent cations in the patch pipette, the channels showed inward rectification in cell-attached patches with a single-channel conductance of 37 pS at negative and 24 pS at positive clamp potentials. 4. With either 70 mM-Ca2+ or Ba2+ in the patch pipette, the current-voltage relation was linear with slope conductances of 16 and 19 pS, respectively. 5. Mean channel open probability increased with increasing pressure and with depolarizing clamp potentials. 6. Cell swelling induced by hypotonic shock activated the SA channels in cell-attached experiments. 7. The SA channel may be involved in cell volume or blood flow regulation. The contribution of these channels to the regulation of cerebrospinal salt and water content, especially in brain oedema, is discussed.

Animals↗
PubMed1992

Characterization of the sodium current in single human atrial myocytes.

Patch-clamp recording techniques have permitted measurement of the fast Na+ current (INa) in isolated cardiac cells from a number of species in recent years. However, there is still only very little information concerning human cardiac INa. The purpose of this study was to describe the kinetics of INa in normal-appearing, Ca(2+)-tolerant, enzymatically isolated human atrial myocytes using whole-cell voltage-clamp techniques. Atrial specimens were obtained from 46 patients undergoing open heart surgery. Cs+ was substituted for K+ in both pipette and external solutions and F- was added to the former. The reversal potential of the rapid inward current varied approximately 57 mV at 17 +/- 1 degrees C with a 10-fold change in [Na+]o, and the current was completely blocked by 100 microM tetrodotoxin, findings typical of the fast cardiac Na+ current. The tetrodotoxin dose-response curve was best fitted by an equation describing binding to high- and low-affinity sites. INa was activated at a voltage threshold of -70 to -60 mV, and peak inward current was obtained at approximately -30 mV (holding potential, -140 mV). The inactivation time course was voltage dependent and was fitted best by the sum of two exponentials. The relation between voltage and steady-state availability (h infinity) was sigmoidal with the half-inactivation at -95.8 +/- 0.9 mV and a slope factor of 5.3 +/- 0.1 mV (n = 46), and we did not observe a significant difference with disease and age. The overlap of the h infinity and activation curves suggested the presence of a Na+ "window" current. Recovery from inactivation also was voltage dependent and best fitted by a model describing the sum of two exponentials. Recovery occurred after an initial delay at potentials positive to -140 mV, suggesting that inactivation of human atrial INa is a multistate process. We conclude that INa of normal-appearing, Ca(2+)-tolerant human atrial myocytes is similar to that of other mammalian cardiac cells with the possible exception of having two tetrodotoxin binding sites.

Adolescent↗
PubMed1992

An electrophysiological characterization of ciliated olfactory receptor cells of the coho salmon Oncorhynchus kisutch.

Electrical properties of ciliated olfactory receptor cells isolated from coho salmon (Oncorhynchus kisutch) were studied using the whole-cell mode of the patch-clamp recording technique. 1. Voltage-dependent currents could be separated into two inward and three outward conductances, including a Na+ current, Ca2+ current and three K+ currents. 2. The components of the outward current varied with the life stage of the salmon from which cells had been isolated. In cells isolated from juvenile fish (parr), a Ca(2+)-dependent K+ current dominated the outward current, whereas in cells isolated from older fish (i.e. fish that had undergone smoltification), a transient K+ current became prominent. 3. Differences in response characteristics of outward currents to internal dialysis with cyclic GMP (but not cyclic AMP) were also correlated to the life stage of salmon. Under conditions in which the Ca(2+)-activated current was blocked, relaxation of the outward current was slowed by dialysis with cyclic GMP only in cells isolated from smolts and sea-run fish, but not in those isolated from mature spawners. 4. From these results, we suggest that hormone modulation of olfactory receptor cell development or differentiation may play a role in establishing these differences.

Animals↗
PubMed1992

Structural and functional crosstalk between acetylcholine receptor and its membrane environment.

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or "annular" lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells.

Acetylcholine↗
PubMed1992

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.

Animals↗
PubMed1992

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.

One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.

Adrenal Glands↗
PubMed1992

Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.

Calcimycin↗
PubMed1992

Multiple histamine receptors: properties and functional characteristics.

Recent advances in molecular biological techniques have seen the cloning of the H2-receptor gene from canine parietal cells, and the structural identification and cloning of the H1- and H3-receptors should soon follow. This information will allow the elucidation of the mechanisms responsible for the species and tissue heterogeneity in the H1-receptor binding and functional characteristics. The mechanisms underlying the inhibitory effect of H3-receptor stimulation on neurotransmitter release remains to be established, but this receptor appears to be coupled to its effector system (perhaps an ion channel?) via a G-protein. Patch-clamp studies on histamine receptors in invertebrate neurones have identified a ligand-gated chloride channel at a photoreceptor synapse of the housefly and it remains an intriguing possibility that there is a counterpart in mammalian species.

Animals↗
PubMed1992

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Adenosine Diphosphate Ribose↗
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

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

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↗