Restoration of voltage-dependent antibrain antibody-inhibited calcium current by cyclic adenosine monophosphate

A dual-microelectrode voltage clamp technique was used for recording voltage-dependent calcium current (I_ca) in unidentified neurons isolated fromHelix pomatia. Neither intracellular injection of cyclic adenosine monophosphate (cAMP; 10 nA, 5 min) nor intracellular application of dibutyril-cAMP (dcAMP; 1 mM, 10–20 min) induced a change in normal I_ca or produce a reversible 10–20% reduction in amplitude. Adding S-100 protein fraction antibodies to the external medium led to the onset of calcium-dependent inactivation of I_ca, bringing amplitude of I_ca down to 15±12% of its initial level. Either cAMP or dcAMP then restored inhibited I_ca to 50±11% of its original level. It was found that the effects of cAMP on I_ca of intact neurons depend on level of cytoplasmic Ca²⁺.Institute for Brain Research, All-Union Center for Mental Health Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 247–252, March–April, 1989.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.     

Mechanisms of accelerating decay of cAMP-induced calcium current

The change in the decay rates of a high-threshold calcium current (I_Ca) when a depolarizing pulse is presented was investigated under conditions of an increased level of cyclic adenosine monophosphate (cAMP) in the cytoplasm of isolated snail neurons using an intracellular injection of cAMP or extracellular application of dibutyryl-cAMP. For 20 of the 38 neurons it was found that cAMP reduced the half-life of the fast and slow components of I_Ca decay by 2–2.5 times. The effect did not depend on the test-pulse potential and was reproduced with respect to a high-threshold barium current. In double-pulse experiments it was found that cAMP enhanced the effect of depolarized prepulses (V_c) on the I_Ca tested (I_t). Analysis of the I_t-V_c curve showed that cAMP enhanced both calcium- and potential-dependent inactivation of I_Ca. It was found that when the interval between the pulses changes, cAMP slows the recovery of the calcium channel from calcium-dependent inactivation. The results suggest that cAMP increases the affinity of the Ca²⁺-channel inactivation substrate for Ca²⁺-ions.Brain Institute, Russian Academy of Medical Sciences, Moscow. Translated from Neirofiziologiya, Vol. 24, No. 1, pp. 60–68, January–February, 1992.     

Effects of quinine on calcium current in mollusk neurons

The effects of quinine on the peak amplitude and the decay of calcium currents (I_Ca) were investigated in nonidentified neurons isolated fromHelix pomatia. A concentration of 1×10^–5–5×10^–4 M quinine was found to produce a reversible dose-dependent deceleration in the decline of I_Ca ("lead" effect) and a reversible, slowly evolving dose-dependent reduction in I_Ca amplitude ("lag" effect). A reduction in amplitude down to half control level is observed at a quinine concentration of 6 ×10^–5 M, while the current-voltage relationship of I_Ca shifts by 5–10 mV towards negative potentials. Results show that quinine successfully blocks calcium channels inHelix pomatia neurons.Institute of Brain Research, All-Union Mental Health Research Center, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 413–417, May–June, 1987.     

Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

Stimulation of neuronal calcium conductance by parathyroid hormone

The effects of 10^–10–10^–5 M parathyroid hormone (PTH) on voltage-dependent potassium channels at theHelix pomatia neuronal membrane were investigated in voltage-clamped experiments using intracellular perfusion techniques. The hormone was found to produce a 2-stage effect on calcium current (I_Ca). The initial, brief stage of PTH action consisted of a minor (7–10%) increase in I_Ca and was partially reversible. This was followed by the second (slow) stage, developing for 60–70 min, whereupon level of I_Ca doubled. This hormonal action was not easily reversed and did not occur unless the intracellular solution contained ATP or the hormone was applied after perfusing the cell. Introducing 10 mM EDTA into the perfusate induced a considerable decline in PTH effects. Adding concentrations of 100 and 60 µM of exogenous cAMP and cGMP, respectively, did not imitate the action of this hormone. The first-mentioned effect is thought to be produced by indirect PTH action on channel protein or structures closely associated with the channel and the second by metabolic processes, possibly the phosphoinositide pathway of signal transmission.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Medical Institute, Erevan. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 373–380, May–June, 1990.     

Investigation of the effect of intracellular calcium on the cAMP-mediated increase in the calcium current

The experiments were conducted on intracellularly persfused neurons of the molluskHelix pomatia, in which a serotonin (5-HT)-induced increase in the calcium current (I_Ca), mediated by cAMP, is observed. It was established that desensitization of the cell to the action of 5-HT is due to an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). At [Ca²⁺]_i=10^–7 and 10^–6 M, the effect of 5-HT constituted 60 and 32% of this value in the control (in the presence of 10 mM EGTA in the intracellular solution), respectively; at [Ca²⁺]_i=10^–5 M, there was no effect of 5-HT at all. The addition of the calmodulin antagonist trifluoperazine (5·10^–5 mM) or blockers of phosphodiesterase (5 mM theophylline or 10^–4 M IBMX) to the perfusate sharply weakened the ability of calcium to inhibit the effect of 5-HT; at [Ca²⁺]_i=10^–5 M, in the presence of one of these compounds, the effect constituted 60–70% of its control value. It is concluded that the calcium-calmodulin-dependent phosphodiesterase is a key link in the interaction of the two-signal transduction pathway — Ca²⁺ and cAMP in modulation of the calcium-channel activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 306–313, May–June, 1991.     

Responses of rat spinal ganglion neurons mediated by activation of serotonin receptors of the third type

Application of serotonin (5-hydroxytryptamine; 5-HT) to rat dorsal root ganglion neurons under conditions in which potassium conductance was blocked by cesium ions elicited depolarizing responses followed by an increase in membrane conductance. The responses did not exhibit desensitization and were due to activation of 5-HT receptors of the third type (5-HT₃Rs), since they were insensitive to methysergide, the 5-HT₂R antagonist, but were inhibited by tropicetrone (ISC 205–930) and metoclopramide, the 5-HT₃R antagonists. The reversal potential of the 5-HT-induced depolarizing responses was –11.9 mV; their amplitude decreased following a decrease in extracellular Na⁺ concentration but remained constant after intracellular injection of GTP. The amplitude of the responses increased following elevation of intracellular cAMP concentration caused by theophylline or sodium fluoride whose potentiating effect was reduced by butamide, a protein kinase A inhibitor. Potentiation of the 5-HT-induced responses was also produced by increased intracellular Ca²⁺ concentration following either direct intracellular injections or a burst of action potentials. The potentiation could be prevented by trifluoroperazine, the calmodulin inhibitor. The 5-HT effects were also potentiated by methylfurmetide, an activator of muscarinic acetylcholine receptors. The effect of methylfurmetide was slightly decreased by trifluoroperazine and was markedly decreased by polymixin B, a protein kinase C inhibitor. The effects of 5-HT were also enhanced by ethanol.Neirofiziologiya/Neurophysiology, Vol. 25, pp. 258–263, July–August, 1993.     

Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Regulation of calcium current through L-type calcium channels (I _Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI _Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI _Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI _Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI _Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI _Ca,L. 8-Bromo-cAMP (1 μM) increasedI _Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI _Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI _Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI _Ca,L (probably through activation of PKA). PKG apparently mediatesI _Ca,L drops evoked by cAMP at high concentrations and by cGMP.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Regulation of the slow Ca++ channels of myocardial cells

Contraction of the heart is regulated by a number of mechanisms, such as neurotransmitters, hormones, autacoids, pH, intracellular ATP, and Ca⁺⁺ ions. These actions are mediated, at least in part, by actions on the sarcolemmal slow (L-type) Ca⁺⁺ channels, exerted directly or indirectly. The major mechanisms for the regulation of the slow Ca⁺⁺ channels of myocardial cells includes the following. cAMP/PK-A phosphorylation stimulates the slow Ca` channel activity, whereas cGMP/PK-G phosphorylation inhibits. DAG/PK-C phosphorylation and tyrosine kinase phosphorylation are suggested to stimulate the slow Ca⁺⁺ channel activity. Intracellular application of G_s protein increases the slow Ca⁺⁺ currents (ICa(L)). Lowering of intracellular ATP inhibits I_Ca(L). Acidosis and increase in [Ca]_i inhibits I_Ca(L). A number of changes in the Ca⁺⁺ channels also occur during development and aging. Thus, it appears that the slow Ca⁺⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors, and thereby control can be exercised over the force of contraction of the heart.     

The effects of cAMP,Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses inHelix neurons

1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.     

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10^–9–10^–8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (I_k(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 356–361, May–June, 1987.     

Tolbutamide enhances potential-dependent inactivation of calcium channels in snail neurons

A two-microelectrode voltage-clamp method was used to measure a high-threshold calcium current (I_Ca) on isolated snail neurons. Tolbutamide (1–5 mmole/liter) and H-8 (1–30 µmole/liter), inhibitors of kinase A, caused a decrease in the peak amplitude and accelerated I_Ca decay during a depolarizing stimulus. The half-life of the "slow" time constant of I_Ca decay decreased in the presence of tolbutamide, and was two to three times stronger than the half-life of the "fast" time constant. I_Ca inactivation curves plotted in a double-stimulus experiment have shown that after tolbutamide application, I_Ca inactivation elicited by the application of high-amplitude depolarizing pre-stimuli preferentially rises (V_c=+30 to +70 mV). The results suggest that dephosphorylation of Ca²⁺ channels enhances a potential-dependent component of the inactivation process.Scientific-Research Institute of the Brain, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 515–519, September–October, 1991.     

The h-Current in the Substantia Nigra pars Compacta Neurons: A Re-examination

The properties of the hyperpolarization-activated cation current (I_h) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I_h kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of −77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on I_h amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the I_h amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in I_h inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of I_h by endogenously released neurotransmitters acting on metabotropic receptors –mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.     

Depressing action of nitroglycerin on voltage-activated calcium current in isolated smooth muscle cells of the guinea pig gut

The effect of nitroglycerin (NG) on inward voltage-activated calcium current (I _Ca) was studied in isolated smooth muscle cells (SMC) of the guinea pigtaenia coli with the voltage clamp technique in an intracellular dialysis mode. Addition of NG (10^–7 to 10^–4 M) to the extracellular solution reduced theI _Ca amplitude and increased theI _Ca inactivation rate. The maximum inhibition ofI _Ca (on the average, by 41.7 ± 4.8%,n=13) was produced by 10^–4 M NG; the inhibition was dose-dependent. No shift of theI _Ca current-voltage curve under the NG influence was observed. Application of dibutyril-cGMP (2·10^–4 M), a membrane-penetrating analog of cyclic 3,5-guanosine monophosphate (cGMP), likewise decreased theI _Ca amplitude and increased its inactivation rate. The results obtained suggest that the NG inhibitory effect onI _Ca is related to a cGMP-dependent modulation of the voltage-activated calcium channels of L-type in the SMC membrane in the guinea pigtaenia coli.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 218–222, May–June, 1994.     

Effects of microiontophoretically injected AMP and cAMP on calcium current in dialyzed Helix pomatia neurons

The effects of injecting cells with adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) on calcium current were investigated during intracellular dialysis ofHelix pomatia neurons. Microiontophoretically injected AMP was found to lead to reinstatement of calcium current following dialysis-induced wash-out, as well as considerable stabilization of this current with the extracellular medium at normal pH. Current-voltage relationship of the current would then undergo a 10 mV shift towards depolarization values. Perfusing the cell with a solution containing 10 mM AMP then produced a qualitatively identical effect. Injecting the neuron iontophoretically with cAMP led to a decline in the amplitude of calcium current under the same conditions. Neither raising the pH of the intracellular solution to 8.1 nor adding 4-aminopyridine in order to depress the hydrogen ion current produced a qualitative alteration in the effects of injecting AMP and cAMP on calcium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 769–776, November–December, 1988.     

Carbonyl stress: malondialdehyde induces damage on rat hippocampal neurons by disturbance of Ca<Superscript>2+</Superscript> homeostasis

The objective of this study was to investigate the influences of carbonyl stress induced by malondialdehyde (MDA), a typical intermediate of lipid peroxidation, on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) alterations in cultured hippocampal neurons of rat. The microphotographic study clearly demonstrated that the hippocampal neurons became gradually damaged following exposure to different concentrations of MDA. Further study indicated that the plasma membrane Ca²⁺-ATPase (PMCA) activity was inhibited by MDA in a concentration- and time-dependent manner. The supplementation of 100 μM MDA was found to cause a notable early phase increase of [Ca²⁺]_i in hippocampal neuron cultures followed by a more pronounced late-phase elevation of [Ca²⁺]_i. Such effect of MDA was prevented by the addition of nimodipine, an inhibitor of L-type calcium channel or by an extracellular Ca²⁺ chelator EGTA. The identification of the calcium signalling pathways were studied by applying U73122, an inhibitor of PL-C, and H-89, an inhibitor of protein kinase A (PKA), showing the involvement of PL-C/IP3 pathway but not the PKA/cAMP pathway. These results suggested that MDA-related carbonyl stress caused damages of rat hippocampal neurons by triggering Ca²⁺ influx and influencing Ca²⁺ homeostasis in cultured neurons, and also MDA may act as a signalling molecule regulating Ca²⁺ release from intracellular stores.     

A Cyclic Nucleotide-dependent Chloride Conductance in Olfactory Receptor Neurons

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca²⁺ as well as in the presence of 3 mm intracellular Ca²⁺, suggesting that the current did not require either voltage or Ca²⁺ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides. Received: 12 November 1996/Revised: 4 April 1997