Reduction Of Calcium Current Research Articles

Modulation of calcium current, intracellular calcium levels and cell survival by glucose deprivation and growth factors in hippocampal neurons

Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) can protect CNS neurons against ischemic/excitotoxic insults, but the mechanism of action is unknown. Imaging of the calcium indicator dye fura-3 and whole-cell patch clamp recordings of calcium currents were used to examine the mechanisms whereby hypoglycemia damages and growth factors protect cultured rat hippocampal neurons. When cultures were deprived of glucose, massive neuronal death occured 16–24 h following the onset of hypoglycemia. Early hypoglycemia-induced changes included calcium current inhibition and a reduction in intracellular free calcium levels ([Ca 2+] i) without morphological signs of neuronal damage. Later changes included a large elevation of [Ca 2+] i which was causally involved in neuronal damage. NGF and bFGF prevented or reduced both early and later responses to hypoglycemia. The growth factors increased calcium (barium) current and [Ca 2+] i to normal limits during the early stages of hypoglycemia and prevented the later elevation in [Ca 2+] i and neuronal damage. Nifedipine, but not omega-conotoxin, blocked calcium currents. The increased calcium current caused by the growth factors was apparently not sufficient to protect neurons against hypoglycemic damage since K + depolarization during the early stages of hypoglycemia did not prevent and, in fact exacerbated, the subsequent neuronal damage. In addition, exposure of neurons to K +, NGF or bFGF only during the first 1 h of hypoglycemia did not protect against hypoglycemic damage. Taken together, the data suggest that neurons initially respond to hypoglycemia with a reduction in calcium currents which may provide a means to maintain [Ca 2+] i within a concentration range conducive to cell survival. Prolonged energy deprivation eventually results in a failure of calcium extrusion systems, glutamate receptor activation and a loss of neuronal calcium homeostasis. Taken together, the data indicate that the mechanism of growth factor protection against energydeprivation involves of the late prevention rise in [Ca 2+] i.

Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells.

The psychoactive properties of Cannabis sativa and its major biologically active constituent, delta 9-tetrahydrocannabinol, have been known for years. The recent identification and cloning of a specific cannabinoid receptor suggest that cannabinoids mimic endogenous compounds affecting neural signals for mood, memory, movement, and pain. Using whole-cell voltage clamp and the cannabinomimetic aminoalkylindole WIN 55,212-2, we have found that cannabinoid receptor activation reduces the amplitude of voltage-gated calcium currents in the neuroblastoma-glioma cell line NG108-15. The inhibition is potent, being half-maximal at less than 10 nM, and reversible. The inactive enantiomer, WIN 55,212-3, does not reduce calcium currents even at 1 microM. Of the several types of calcium currents in NG108-15 cells, cannabinoids predominantly inhibit an omega-conotoxin-sensitive, high-voltage-activated calcium current. Inhibition was blocked by incubation with pertussis toxin but was not altered by prior treatment with hydrolysis-resistant cAMP analogues together with a phosphodiesterase inhibitor, suggesting that the transduction pathway between the cannabinoid receptor and calcium channel involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. However, the development of inhibition is considerably slower than a pharmacologically similar pathway used by an alpha 2-adrenergic receptor in these cells. Our results suggest that inhibition of N-type calcium channels, which could decrease excitability and neurotransmitter release, may underlie some of the psychoactive effects of cannabinoids.

Differential G protein-mediated coupling of D 2 dopamine receptors to K + and Ca 2+ currents in rat anterior pituitary cells

In anterior pituitary cells, dopamine, acting on D 2 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 D 2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either G oα, G 3α, or G i1,2α. Dialysis with G oα antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by G i3α antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D 2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Divalent ions released from stainless steel hypodermic needles reduce neuronal calcium currents

We have investigated the effects of saline solutions exposed to stainless steel hypodermic needles on their ability to reduce voltage-dependent calcium currents in chick dorsal root ganglion neurons and rat cerebellar Purkinje cells. Salines exposed to needles with brass hubs, but not those with plastic hubs, for as little as 2-3 sec reduced calcium currents in both cell types. The amplitude of the response was exposure-dependent and reversible. Elemental analysis of the exposed salines using inductively-coupled-plasma atomic emission spectroscopy revealed that Cu and Zn (but not Cd, Cr, Co, Fe, Mn, Ni or Pb) were released from the brazed needles. The amount of Cu plus Zn released in 30 sec was estimated to be 12-26 microM, depending upon the specific needle examined. Longer exposures produced proportionately higher concentrations of the metals. Dose-response curves for Cu or Zn ions applied directly to cells confirmed that similar concentrations of these ions reduced neuronal calcium currents. Our results indicate that divalent ions released from stainless steel hypodermic needles with brass hubs can interfere with measurements of calcium currents. In addition the results contribute new information regarding potential physiological and pathological actions of copper and zinc ions in biological tissues.

Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium.

We investigated the effects of 1 and 3 mM 2,3-butanedione monoxime (BDM, diacetyl monoxime) on excitation and contraction of cardiac muscle in several types of preparations at various levels of organization. We selected a concentration of BDM that was not expected to affect sarcolemmal calcium flux and action potential duration in cardiac tissue. Two indicators were used to record intracellular calcium. Aequorin, a bioluminescent calcium indicator, was used in studies with ferret papillary muscle preparations, and fura-2, a fluorescent calcium indicator, was used in studies with guinea pig cardiac myocytes. In both cases, addition of BDM resulted in a reduction of peak intracellular calcium released from the sarcoplasmic reticulum and a reduction of peak twitch force. The duration of the action potential of isolated myocytes was slightly abbreviated in the presence of BDM. In studies on the calcium current in the myocytes, addition of BDM was associated with reduced calcium current at any potential. Peak calcium current was reduced by 7.9 +/- 1% in the presence of BDM. In tetanized ferret papillary muscles, BDM reduced maximal calcium-activated force by 30 +/- 5% and increased the calcium ion concentration required for half-maximal force by 0.1 +/- 0.01 microM. The Hill coefficient was reduced from 5.00 +/- 0.11 to 3.40 +/- 0.20. Maximal shortening velocity of ferret papillary muscles was increased in the presence of BDM from 1.55 +/- 0.24 to 2.04 +/- 0.33 mm/sec. Ca2+ binding to troponin C in skinned fiber preparations from guinea pig, bovine, and canine hearts was unaffected by addition of up to 10 mM BDM. Our results indicate that BDM affects both calcium availability and responsiveness of the myofilaments to Ca2+. Uncoupling of contractile activation from excitation may also result from altered crossbridge kinetics.

Halothane reduces calcium currents in clonal (GH3) pituitary cells.

Halothane reduces calcium currents in clonal (GH3) pituitary cells.

The reduction of neuronal calcium currents by ATP-γ-S is mediated a G protein and occurs independently of cyclic AMP-dependent protein kinase

We studied the effects of ATP-γ-S on the T, N and L calcium current components of nodose ganglion neurons using the whole cell variation of the patch clamp technique. ATP-γ-S can serve as a phosphate donor in kinase-mediated reactions, the donated phosphate group being resistant to the action of phosphateses. We therefore compared the effect of ATP-γ-S to that of the catalytic subunit of the cyclic AMP-dependent protein kinase (AK-C), included in the recording pipette with 5 mM ATP. AK-C (50 μg/ml) had no effect on the T current, and caused a∼30% increase in currents containing the N and L components during a 20-min recording, as compared to a∼45% decrease in control currents. In contrast, in the presence of 2.5 mM ATP-γ-S, T currents declined∼30%, and currents containing the N and L components declined to a greater extent than control currents, about 65%. In addition, the time to peak current was increased from∼ 14ms to∼40ms. This effect of ATP-γ-S on calcium currents was similar to that of certain neurotransmitters or GTP-γ-S, an activator of G proteins, except that the effects of ATP-γ-S were delayed 5–7 min relative to GTP-γ-S. The effects of both ATP-γ-S and GTP-γ-S were reduced or abolished in neurons treated with pertussis toxin. We conclude that AK-C regulates neuronal calcium currents, presumably by phosphorylation of channels or associated proteins, and that the ATP-γ-S-induced reduction of calcium currents cannot be due to its serving as a phosphate donor for endogenous AK. Instead, ATP-γ-S reduces calcium currents via a G protein, perhaps by its conversion to GTP-γ-S.

Dynorphin A and cAMP-dependent protein kinase independently regulate neuronal calcium currents.

The kappa-selective opioid peptide dynorphin A (DYN) inhibits neuronal adenylate cyclase activity and reduces neuronal voltage-dependent calcium currents. It is not yet known, however, whether the regulation of calcium channel activity is dependent on or independent of the adenylate cyclase/cAMP system. We used the whole-cell variation of the patch clamp technique to show that DYN reversibly reduced, in a naloxone-sensitive manner, calcium currents in acutely dissociated rat nodose ganglion neurons. DYN slowed the rate of current activation and had a greater effect on currents evoked from relatively negative holding potentials. These actions were mimicked by guanosine 5'-[gamma-thio]triphosphate, which activates GTP-binding proteins (G proteins), and were blocked by pretreatment with pertussis toxin, which inactivates Gi- and Go-type G proteins. In contrast, calcium currents recorded in the presence of the catalytic subunit of the cAMP-dependent protein kinase (AK-C), included in the recording pipette, increased in magnitude throughout the recording. DYN was applied to neurons before and after the effect of AK-C became apparent; the reduction of calcium currents by DYN was greater in the presence of AK-C than in its absence. We conclude that the acute reduction of neuronal calcium currents by DYN occurred by means of activation of pertussis toxin-sensitive Gi- or Go-type G proteins. The persistence of the action of DYN in the presence of AK-C indicates, however, that this effect was independent of a reduction of the activity of the adenylate cyclase/cAMP system and suggests in addition that phosphorylated channels may be preferentially inhibited by DYN.

LHRH and GTP-γ-S modify calcium current activation in bullfrog sympathetic neurons

LHRH (chicken II luteinizing hormone-releasing hormone) partially reduces calcium currents and slows the activation kinetics of part of the remaining current in frog sympathetic neurons. The effects of LHRH are mimicked by intracellular dialysis with GTP-γ-S. A strong depolarization can temporarily reverse the effects of LHRH or GTP-γ-S: activation kinetics return to normal, and the amplitude of the current is increased (facilitation). Facilitation develops rapidly (π = 4–6 ms at >+30 mV) and decays more slowly ( t 1 2 = 60 ms at −80 mV). Tail currents in LHRH are smaller and faster than in the control, and these effects are partially reversed by facilitation. These results can be explained by a model in which a fraction of the channels is shifted into a “reluctant” gating mode, where opening requires stronger depolarization. If this mechanism is at the root of presynaptic inhibition, our results predict that inhibition of transmitter release would be overcome during bursts of high frequency activity.

Neuropeptide Y reduces calcium current and inhibits acetylcholine release in nodose neurons via a pertussis toxin-sensitive mechanism

1. The effect of neuropeptide Y (NPY) on voltage-dependent calcium currents was studied in acutely dissociated rat vagal afferent (nodose) neurons by the use of both intracellular single-electrode and whole-cell patch-clamp techniques. 2. Nodose neurons exhibited three calcium current components similar to the transient low-threshold (T), slowly inactivating high-threshold (L), and the transient high-threshold (N) currents previously described in dorsal root ganglion neurons (Nowycky et al. 1985). The characteristics of calcium current components were similar for the two recording techniques except that the inactivation time constants (tau i) were two- to threefold larger at 22 degrees C (whole-cell patch clamp) than at 35 degrees C (single-electrode voltage clamp). 3. NPY (0.1-100 nM, ED50 4 nM) produced a concentration-dependent reduction in calcium currents with the use of both recording techniques. NPY (100 nM) had no effect on T and L currents but reduced the combined N/L current 31 +/- 6% in 47% of the cells tested. Current traces were also analyzed by multiexponential curve fitting to determine amplitudes and inactivation time constants (tau i). NPY selectively reduced the amplitude of the curve-fitted N current component 45 +/- 8% but had no effect on any of the tau i. The effect of NPY to reduce calcium current was blocked in the presence of gadolinium (1 microM), a putative N channel antagonist. Pretreatment of cultures with pertussis toxin (PTX) (100 ng/ml) for 16-24 h blocked the effect of NPY. 4. NPY reduced the peak current without changing the voltage dependence of the peak current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)

2‐Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin‐sensitive manner.

1. The adenosine analogue 2-chloroadenosine (CADO) reduced the duration of calcium-dependent action potentials (CAPs) in mouse dorsal root ganglion (DRG) neurones in culture, by reducing voltage-activated calcium conductance (Macdonald, Skerritt & Werz, 1986). Using the single-electrode voltage clamp technique, we recorded three calcium current components in these neurones, the transient low-threshold (T), transient high-threshold (N) and slowly inactivating high-threshold (L) currents, as described previously (Nowycky, Fox & Tsien, 1985; Gross & Macdonald, 1987). CADO (100 microM) had no effect on the isolated T and L currents. In contrast, CADO reduced calcium currents evoked at clamp potentials positive to -20 mV from holding potentials (Vh) near the resting membrane potential; under these conditions, the calcium current consisted primarily of N and L calcium current components. 2. This effect of CADO was not voltage dependent. CADO reduced the magnitude of the calcium current without affecting the voltage dependence of the calcium current-voltage relation. In addition, similar reductions of calcium current were observed when currents were evoked from Vh of -60 or -80 mV. 3. In order to determine if a guanine nucleotide-binding (G) protein was involved in the CADO effect on calcium current, cultures were pre-treated with pertussis toxin (PT) for at least four hours. PT (100 ng/ml) reduced or abolished the CADO-induced reduction of CAP duration and calcium current. 4. Since CADO inhibits adenylate cyclase through the PT-sensitive G protein, Gi, we compared the effects of CADO and 8-Br-adenosine 3',5'-cyclic-monophosphate (8-Br-cyclic AMP) on calcium current. The effect of 8-Br-cyclic AMP was voltage dependent, unlike that of CADO. 8-Br-cyclic AMP reduced calcium currents evoked from Vh = -65 mV, but had no effect on currents evoked from Vh = -85 mV. 5. We conclude that the adenosine agonist CADO reduced CAP duration in mouse DRG neurones by selectively reducing the N current component, and that the coupling between the adenosine receptor and the calcium channel required a PT-sensitive G protein. The CADO effect was unlikely, however, to be due to modulation of adenylate cyclase activity.

Antiepileptic Drug Actions

Antiepileptic drugs (AEDs) vary in their efficacy against generalized tonic-clonic, myoclonic, and absence seizures, suggesting different mechanisms of action. Phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA) reduced the ability of mouse central neurons to sustain high-frequency repetitive firing of action potentials (SRF) at therapeutic free serum concentrations. Phenobarbital (PB) and the benzodiazepines (BZDs), diazepam (DZP), clonazepam (CZP), and lorazepam (LZP), also reduced SRF, but only at supratherapeutic free serum concentrations achieved in treatment of generalized tonic-clonic status epilepticus. These AEDs interact with sodium channels to slow the rate of recovery of the channels from inactivation. The BZDs and PB enhanced gamma-aminobutyric acid (GABA) responses evoked on mouse central neurons by binding to two different sites on the GABAA receptor channel. BZDs increased the frequency of GABA receptor channel openings. In contrast, barbiturates increased the open duration of these channels. VPA enhanced brain GABA concentration and may enhance release of GABA from nerve terminals. Ethosuximide (ESM) reduced a small transient calcium current which has been shown to be involved in slow rhythmic firing of certain neurons. Reduction of SRF, enhancement of GABA-ergic inhibition, and reduction of calcium current may be, in part, the bases for AED action against generalized tonic-clonic, myoclonic, and absence seizures, respectively.

Tamoxifen Reduces Calcium Currents in a Clonal Pituitary Cell Line*

The effect of the anti-estrogen Tamoxifen (Tx) on membrane electrical properties and the underlying calcium (Ca2+) conductances was examined in the clonal pituitary cell line GH3/B6 which exhibits calcium action potentials at rest. Electrophysiological recordings (109 cells) were made using either high resistance intracellular microelectrodes or the whole-cell recording (WCR) patch-clamp technique. Electrical activities of 39 spontaneously active GH3/B6 cells were recorded with sharp microelectrodes filled with 3 M KCl. The spikes were Ca2+-dependent since they were blocked by Cobalt ions (Co2+, 5 mM). When applied directly to the recorded cell, Tx (10(-7) M) inhibited action potential firing. This blockade was accompanied by a discrete hyperpolarization of the membrane potential (-2.8 +/- 2 m V) from rest (-39.5 +/- 5 m V) and a 10% increase in the input membrane resistance. The effect stopped soon after Tx removal (mean 11.4 +/- 6 sec), and Tx solvent was unable to elicit the response. Current clamp WCR with pipettes containing potassium gluconate (KGlu, 140 mM) confirmed these results (30 cells), but the addition of cell extract to KGlu was necessary to prevent rundown of the response and to obtain reproducible action potential blockade. Short (1-5 min) or long term (48 h) pretreatment with estradiol (10(-7) to 10(-5) M) did not change the response to Tx, thus indicating that its effect was not mediated through estrogen receptors. Voltage clamp WCR study of the effect of Tx (10(-7) M) using pipettes filled with cesium chloride (140 mM) showed that both fast and slow inactivating calcium conductances were inhibited (38 cells). The fast inactivating Ca2+ current was reduced by about 60-80% whereas slow inactivating Ca2+ current was completely inhibited. This action may represent one way by which the antitumoral effect of this antiestrogen is mediated.

Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture.

Opioid receptors are differentially coupled to ion channels. Mu- and delta-opioid receptors are coupled to calcium- and/or voltage-dependent potassium channels and kappa-opioid receptors are coupled to voltage-dependent calcium channels. Using the single-electrode voltage-clamp technique, we investigated the effect of the kappa-opioid receptor agonist dynorphin A on somatic calcium currents of mouse dorsal root ganglion (DRG) neurons in culture. Three different calcium currents were recorded: a small transient current activated positive to -60 mV; a large, inactivating current activated positive to -50 mV; and a moderate, slowly inactivating current activated positive to -40 mV. The first was less sensitive to cadmium block than the others. These calcium currents were similar to those described in other cells, which have been designated T, N, and L calcium currents, respectively. The opioid peptide dynorphin A reduced calcium current by selectively reducing the large inactivating (N) calcium current. Naloxone, an opioid receptor antagonist, reversed this action of dynorphin A. N calcium current is the predominant calcium current in DRG neurons. If N calcium channels are present in primary afferent terminals, and if they are coupled to kappa-opioid receptors as in the soma, these results suggest a mechanism by which dynorphin A inhibits calcium influx and neurotransmitter release.

Effects of trimetazidine on action potentials and membrane currents of guinea-pig ventricular myocytes

The effects of trimetazidine on membrane potentials and membrane currents of enzymatically isolated guinea-pig ventricular cells were studied with the use of giga-seal suction pipettes for patch clamp. Trimetazidine (3 X 10(-5) M) decreased the action potential duration from 433 +/- 179 ms (mean and S.D., n = 9) to 319 +/- 156 ms within 8 mins. In voltage clamp experiment, trimetazidine at a concentration of 1.5 X 10(-4) M decreased the peak amplitude of calcium current by 40% (0.92 +/- 0.46 nA to 0.55 +/- 0.19 nA, mean +/- S.D., n = 5). The effect on calcium current was rate-dependent, e.g., at 1 Hz, trimetazidine blocked a larger fraction of the calcium current than at 0.2 Hz. The drug decreased the conductance of potassium current which flows via inward rectifier potassium channel from 28 +/- 11 nS to 19 +/- 10 nS, n = 5, P less than 0.05). Trimetazidine shifted the steady state current-voltage relationship outward at potentials positive to -20 mV. This shift was not due to the enhanced time- and voltage-dependent outward current (Ik). From these findings, it was concluded that trimetazidine shortens action potential duration by blocking the calcium channels with increases in steady state outward current or a possible blockade of non-inactivated component of the calcium current, at the plateau potentials. The reduction of calcium current and of inward rectifier potassium current may protect the cardiac cells from accumulation of calcium ions and from loss of potassium ions, in the presence of ischemia.

The reduction of calcium current in the early embryos of the mouse and the hamster

The reduction of calcium current in the early embryos of the mouse and the hamster

The reduction of calcium current in the early embryos of the mouse and the hamster

Gene expression in rabbit early development was investigated by microinjecting LacZ DNA and LacZ RNA in 1-cell and 2-cell embryos. Expression of LacZ DNA could not be obtained before 30–36 hpf, although synthetic LacZ RNA was translated from 12 hpf at the least. The onset of expression of microinjected DNA correlated with the 8- to 16-cell stage. This suggests that before this stage, there is a general negative control of gene expression. The arrest of in vitro development at the 2- to 8-cell stages did not inhibit LacZ expression, which still occurred at 33 hpf. In addition the inhibition of the first cleavage by nocodazole resulted in LacZ expression in 1-cell embryos. Expression of microinjected DNA thus occurs at a fixed time after fertilization and is independent of cleavages and of the second and subsequent DNA replications. Therefore, the changes in permissiveness for the expression of microinjected DNA in rabbit embryos are reminiscent of those in mouse embryos. Transcriptional selectivity in rabbit embryos was compared to that in early mouse embryos. In both species, Sp1-sensitive promoters were active and the promoter of simian virus 40 did not require far upstream enhancers before late cleavage stages; genes driven by the −447, +563 region of murine leukemia virus were repressed. In rabbit, however, the H-2Kb promoter active in mouse was silent. Altogether, the results illustrate a remarkable conservation of the characteristics of the transcription in early rabbit and mouse embryos and the independence of its resumption from the pattern of cleavage.

Reduction of calcium currents in identified neurons of Helix pomatia: Intracellular injection of D890

1. 1. The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. 2. 2. The TTX-resistant component of the inward current, the inward currents in Na +-free sucrose solution and in Ca 2+ -free Ba 2+ solution were reduced. 3. 3. In Ca 2+-free Co 2+ solution the inward current was not affected. 4. 4. The late outward currents were strongly reduced. In solutions containing 20 mmol/1 NiCl 2 the1 remaining parts of these currents were blocked only to a lesser extent. 5. 5. The early outward current remained unchanged. 6. 6. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.

Calcium entry leads to inactivation of calcium channel in Paramecium.

Under depolarizing voltage clamp of Paramecium an inward calcium current developed and subsequently relaxed within 10 milliseconds. The relaxation was substantially slowed when most of the extracellular calcium was replaced by either strontium or barium. Evidence is presented that the relaxation is not accounted for by a drop in electromotive force acting on calcium, or by activation of a delayed potassium current. Relaxation of the current must, therefore, result from an inactivation of the calcium channel. This inactivation persisted after a pulse, as manifested by a reduced calcium current during subsequent depolarization. Inactivation was retarded by procedures that reduce net entry of calcium, and was independent of membrane potential. The calcium channel undergoes inactivation as a consequence of calcium entry during depolarization. In this respect, inactivation of the calcium channel departs qualitatively from the behavior described in the Hodgkin-Huxley model of the sodium channel.