Intracellular Injection Of Cyclic AMP Research Articles

Neuron quantum computers and a way to unification of science: A compendium of Efim Liberman's scientific work

In 1972, Efim Liberman, a Soviet biophysicist, pioneered a brand-new approach to studying the operation of the brain, the live cell and the human mind by publishing a paper titled “Cell as a molecular computer” (1972). In this paper, Liberman posited that a consecutive/parallel stochastic molecular computer (MCC) controls a living cell. An MCC operates with molecule-words (DNA, RNA, proteins) according to the program recorded in DNA and RNA. Computational operations are implemented by molecular operators acting as enzymes. An MCC is present in each live cell. A neuron cell MCC can be involved in solving tasks for the entire organism. Neuron MCC investigation was started with studying an impact of an intracellular injection of cyclic AMP on electric activity of a neuron. Cyclic nucleotides were considered as input words for an MCC, which are generated inside a neuron as a result of synaptic activity. This led Efim Liberman to the idea that, in order to solve complex physical problems, which are encountered by a neuron and require rapid solutions, the molecular computer adjusts the operation of the quantum molecular regulator, which uses the “computational environment” of the cytoskeleton and quantum properties of the elementary hypersound quasiparticles for completing mathematical operations for the minimum price of action. Efim Liberman suggested that the human self-consciousness is a quantum computer of even a higher level and designated it as an extreme quantum regulator. In order to describe such systems, he suggested to join biology, physics and mathematics into a unified science, and formulated its four fundamental principles. Results of Efim Liberman’s theoretical and experimental studies on the topic of biological computation are summarized in this review.

INTRACELLULAR CYCLIC-AMP SUPPRESSES THE PERMEABILITY OF GAP JUNCTIONS BETWEEN RETINAL AMACRINE CELLS

Gap junctions are intercellular channels composed of subunit protein connexin and subserve electrotonic transmission between connected neurons. Retinal amacrine cells, as well as horizontal cells of the same class, are homologously connected by gap junctions. The gap junctions between these neurons extend their receptive fields, and may increase the inhibitory postsynaptic effects in the retina. In the present study, we investigated whether gap junctions between the neurons are modulated by internal messengers. The permeability of gap junctions was examined by the diffusion of intracellularly injected biotinylated tracers, biocytin or Neurobiotin, into neighboring cells since gap junctions are permeable to these molecules freely. 4% Lucifer Yellow and 6% biocytin or Neurobiotin were injected intracellularly into horizontal cells and amacrine cells in isolated retinas of carp and goldfish and Japanese dace following electrophysiological identification. In the control condition, the tracer spread into many neighboring cells from the recorded cells. Superfusion of retinas with dopamine (100 microM) suppressed diffusion of the tracer into the neighboring horizontal cells, but not in the case of amacrine cells. Intracellular injection of cyclic AMP (300 mM) completely blocked diffusion of the tracer into neighboring horizontal cells and amacrine cells. However, superfusion of retinas with 8-bromo-cyclic AMP (2 mM), membrane permeable cyclic AMP analog, permitted the tracer to diffuse into the neighboring horizontal cells or amacrine cells. Intracellular injection of cyclic GMP (300 mM) blocked the diffusion between neighboring horizontal cells, but did not suppress the diffusion between amacrine cells. These results show that the permeability of gap junctions between amacrine cells is regulated by high concentration of intracellular cyclic AMP level, but not for intracellular cyclic GMP or applied dopamine or extracellularly applied low concentrations of intracellular cyclic AMP level. The present study suggests that these laterally oriented inhibitory interneurons, horizontal cells and amacrine cells, express different connexins which may be differentially regulated by intercellular messengers.

Differential modulation of voltage‐activated conductances by intracellular and extracellular cyclic nucleotides in leech salivary glands

1. Two-electrode voltage clamp was used to study the effects of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) on voltage-dependent ion channels in salivary gland cells of the leech, Haementeria ghilianii. 2. Intracellular cyclic AMP specifically blocked delayed rectifier K+ channels. This was shown by use of 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), forskolin (an activator of adenylyl cyclase) and intracellular injection of cyclic AMP and its dibutyryl and 8-bromo analogues. Cyclic AMP appeared to be the second messenger for the putative neuroglandular transmitter, 5-hydroxytryptamine. 3. Intracellular injection of cyclic GMP specifically potentiated high-voltage-activated (HVA) Ca2+ current and the effect was mimicked by zaprinast, an inhibitor of cyclic GMP-dependent phosphodiesterase. 4. Extracellularly, cyclic GMP and cyclic AMP specifically decreased the amplitude and increased the rate of inactivation of HVA Ca2+ current. These effects of the cyclic nucleotides are identical to those known for extracellular ATP, which activates a presumed purinoceptor. The pyrimidine nucleotide, UTP, was almost equipotent to ATP (threshold dose < 10(-6) M), indicative of a vertebrate-type nucleotide receptor. However, suramin (5 x 10(-5) M), a non-specific P2-receptor antagonist, failed to block the effects of 5 x 10(-6) M ATP (higher suramin doses could not be reliably tested because of the depolarization and increase in membrane conductance produced by the drug). 5. Activation of the putative purinoceptor by ATP did not affect inward rectifier Na+/K+ current which is known to be potentiated by intracellular cyclic AMP and reduced by intracellular cyclic GMP. 6. The preparation may provide a useful model for study of nucleotide actions, and interactions, in channel modulation. It has technical advantages such as large cells (1200 microns in diameter) which lack intercellular coupling and may be individually dissected for biochemical studies.

Intracellular injection of cyclic AMP inhibits the dopamine-induced K(+)-dependent responses of Aplysia ganglion cells.

Intracellular injection of cyclic AMP inhibits the dopamine-induced K(+)-dependent responses of Aplysia ganglion cells.

Caffeine inhibits inositol trisphosphate‐mediated liberation of intracellular calcium in Xenopus oocytes.

1. Voltage-clamp recording of Ca(2+)-activated chloride currents in Xenopus oocytes was used to study the effects of caffeine on the liberation of intracellular Ca2+ induced by photo-release of inositol 1,4,5-trisphosphate (InsP3) from caged InsP3. Bath application of caffeine, at concentrations between 0.1 and 10 mM, reduced or abolished the current evoked by photo-release of InsP3 and by microinjection of InsP3. 2. Caffeine did not appreciably reduce currents evoked by injection of Ca2+ into oocytes, whereas measurements using the Ca2+ indicator Rhod-2 showed that it instead inhibited the liberation of Ca2+ by InsP3. 3. Caffeine increased the threshold amount of InsP3 required to evoke a current response and proportionally reduced the currents evoked by suprathreshold levels of InsP3. 4. Theophylline and 3-isobutyl-1-methylxanthine (IBMX) were much less potent than caffeine, and few changes were seen in the InsP3 responses following application of forskolin or intracellular injection of cyclic AMP. Thus, inhibition of InsP3 responses by caffeine does not arise through inhibition of phosphodiesterase enzymes. 5. Even at high (10 mM) concentrations, caffeine did not itself elicit any clear Ca(2+)-activated current. It is therefore unlikely that inhibition of the InsP3 responses arise because caffeine itself liberates Ca2+ from intracellular stores. 6. The site of action of caffeine is intracellular, because injections of caffeine into the oocyte strongly inhibited responses to InsP3, whereas local extracellular applications of similar amounts were almost without effect.

Decoupling of horizontal cells in carp and turtle retinae by intracellular injection of cyclic AMP.

1. Horizontal cells are electrically coupled through gap junctions. This is a disadvantage in elucidating the membrane properties of the cells. In order to block gap junctions, adenosine 3',5'-cyclic monophosphate (cyclic AMP) or its analogues, dibutyryl cyclic AMP and 8-bromo cyclic AMP, were ionophoretically injected into horizontal cells of the carp or turtle retina. 2. Before injection of the chemicals the input resistance of the cell was so low as to be unmeasurable, because the applied current leaked through gap junctions. After injection, however, the input resistance was significantly increased. 3. After the injection dye-coupling between horizontal cells was not observed when examined by intracellular injection of Lucifer Yellow dye, supporting the idea that high concentrations of intracellular cyclic AMP block gap junctions. 4. In this situation responses to light delivered to the receptive field centre were increased in amplitude, while responses to light delivered to the receptive field surround were greatly diminished. 5. After injection horizontal cells were readily polarized by conventional intracellular current injection. The hyperpolarizing light responses in carp and turtle luminosity-type cells (H1 cells) could be reversed by depolarizing the horizontal cells, and the reversal potentials were estimated to be about 0 mV. In addition, the resistance increase which accompanied the hyperpolarizing light responses could be detected. 6. In turtle biphasic chromaticity-type horizontal cells (H2 cells), hyperpolarizing light responses to shorter wavelengths and depolarizing ones to longer wavelengths could be reversed by depolarizing the horizontal cells. Both responses have almost the same reversal potential at about 0 mV. The membrane resistance changes associated with light responses were also detected; the resistance increased during the hyperpolarizing response, while it decreased during the depolarizing response. These observations suggest that the ionic mechanisms of both responses are probably the same, irrespective of their polarities.

Action of cyclic AMP on intracellular calcium concentration and bursting activity

In order to clarify its role in the provocation of seizure activity, the effects of cyclic AMP were examined on the intracellular calcium concentration of pentylenetetrazol (PTZ)-sensitive neurons as well as of PTZ-non-sensitive neurons of the Japanese land snail, Euhadra peliomphala. Extracellular application of isobutylmethylxanthine (IBMX) and dibutyryl cyclic AMP showed bursting activity-like firing in the PTZ-sensitive neurons. When 5′-guanylylimidodiphosphate (GNP-PNP) was injected into PTZ-sensitive neurons in the extracellular presence of IBMX and dibutyryl cyclic AMP, bursting activity followed by long-lasting hyperpolarization occurred. Intracellular injection of cyclic AMP into PTZ-sensitive neurons caused hyperpolarization coincident with an increase in intracellular calcium concentration. This increase in intracellular calcium concentration was the same under conditions in which the calcium influx was inhibited by the substitution of extracellular calcium chloride by cobalt chloride. In PTZ-non-sensitive neurons, cyclic AMP-induced bursting activity was not observed. These results suggest that an increase in cyclic AMP provoked bursting activity via an increase in intracellular calcium concentration.

Elevation of adenosine 3',5'‐phosphate mimics slow synaptic excitation in myenteric neurones of the guinea‐pig.

Intracellular recording was used to assess the effects of elevation of intraneuronal adenosine 3',5'-phosphate (cyclic AMP) on electrical behaviour of AH/Type 2 myenteric neurones in guinea-pig small intestine. Intracellular injection of cyclic AMP resulted in membrane depolarization, increased input resistance, suppression of post-spike hyperpolarizing potentials and enhanced excitability. Application of 8-(4-chlorophenylthio) cyclic AMP or 8-bromo cyclic AMP, which are membrane permeant analogues of cyclic AMP, resulted in the same changes in electrical behaviour as intracellular injection of cyclic AMP. Treatment with the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, resulted in the same changes in neuronal electrical behaviour as intracellular injection of cyclic AMP and application of permeable analogues of cyclic AMP. Each of the treatments, which were expected to elevate intraneuronal cyclic AMP, duplicated the electrophysiological behaviour of AH/Type 2 neurones during slow synaptic excitation. The results support the hypothesis that the biochemical basis for slow synaptic excitation in AH/Type 2 myenteric neurones of guinea-pig small intestine is increased synthesis of cyclic AMP in the cell soma.

Opposing roles for D-1 and D-2 dopamine receptors in regulating the excitability of growth hormone-producing cells in the snail Lymnaea stagnalis

Dopamine hyperpolarizes growth hormone-producing cells (GHC) in the CNS of Lymanea stagnalis. This effect of dopamine was mimicked by the D-2 receptor agonist LY 141865 and antagonized by the D-2 receptor antagonists (−) sulpiride and YM 09151-2. SKF 38393, a selective D-1 receptor aagonist, increased the excitability of the GHC. This effect was mimicked by intracellular injection of cyclic AMP andantonized by the D-1 receptor antagonist SCH23390. Dopamine (in the presence of (−)-sulpride) also increased the excitability of the GHC. It is concluded that both a D-1 and a D-2 receptor regulate the electrical activity of the GHC in the CNS of Lymnaea stagnalis.

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.

Serotonin modulates a specific potassium current in the sensory neurons that show presynaptic facilitation in Aplysia.

Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by decreasing outward K+ current. The depression of outward current delays repolarization of the action potential and extends the duration of Ca2+ influx into the presynaptic terminals, thereby contributing to the facilitation of transmitter release that underlies behavioral sensitization. We have extended the analysis of serotonin's action and find that it acts on a specific serotonin-sensitive K+ current (S current), which is different from the early K+ current (IA), the delayed K+ current (IK), the Ca2+-dependent K+ current (IC), and the muscarine-sensitive M current. The serotonin-sensitive current in these cells persists when IA and IK are reduced by conditioning depolarization or channel-blocking agents. The S current is not activated by intracellular injection of Ca2+, nor is it affected by substitution of Ba2+ for Ca2+, a treatment that reduces IC. Moreover, intracellular injection of cyclic AMP exerts an effect indistinguishable from that of serotonin. This observation and the insensitivity of the current to Ba2+ distinguishes the S current from M current. S current is activated at the resting potential and does not inactivate with steady-state depolarization. It is active sufficiently early during an action potential to contribute to the repolarization of the action potential and therefore accounts for the physiological effects of serotonin.

Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones.

We have identified a serotonin-sensitive K+ channel with novel properties. The channel is active at the testing potential; its gating is moderately affected by membrane potential and is not dependent on the activity of intracellular calcium ions. Application of serotonin to the cell body or intracellular injection of cyclic AMP causes prolonged and complete closure of the channel, thereby reducing the effective number of active channels in the membrane. The closure of the channel can account for the increases in the duration of the action potential, Ca2+ influx, and transmitter release which underlie behavioural sensitization, a simple form of learning.

Does cyclic 3' ,5'-adenosine monophosphate act as second messenger in a voltage-dependent response to 5-hydroxytryptamine in Aplysia?

1 The possibility that cyclic adenosine 3',5'-monophosphate (cyclic AMP) mediates a voltage-dependent inward current elicited by 5-hydroxytryptamine (5-HT) in RB and LB cells of the abdominal ganglion of Aplysia was tested. 2 Intracellular injection of cyclic AMP elicited an inward current with a similar time course, potential dependence and ionic sensitivity as the response to 5-HT. 3 Intracellular injection of guanylyl imidodiphosphate (GMP-PNP), which activated adenylate cyclase, neither mimicked nor enhanced the 5-HT-evoked current. On the contrary, it reduced the current. 4 The phosphodiesterase inhibitors, Ro20-1724, isobutyl methylxanthine (IBMX) and theophylline, each antagonized the voltage-dependent response to 5-HT. To varying degrees they each induced an inward current. 5 The adenylate cyclase antagonist, dithiobisnitrobenzoic acic (DTNB), had no effect on the response to 5-HT when applied either intracellularly or extracellularly. Intracellular injection of the phosphodiesterase activator imidazole also had no effect. 6 Tubocurarine and neostigmine did not reduce the voltage-dependent inward current evoked by 5-HT; methysergide elicited an inward current. 7 Although the observations that cyclic AMP and 5-HT can evoke similar voltage-dependent inward currents in RB and LB neurones of Aplysia might suggest a second messenger role for the cyclic nucleotide, the pharmacological data are inconsistent with this hypothesis.

Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones.

One model of synaptic transmission suggests that transmitters modify postsynaptic permeability through the intermediary of cyclic AMP. Thus, serotonin (5-hydroxytryptamine) evokes in molluscan neurones a decrease in a voltage-dependent K+ conductance which in turn generates a slow inward current when studied in steady voltage-clamp conditions. The serotonin-induced increase of the plateau phase of the spike of an Aplysia sensory neurone can be mimicked by both intracellularly injected cyclic AMP and extracellularly applied phosphodiesterase inhibitors, suggesting that cyclic AMP mediates the effect. We have tested whether a similar mechanism could account for the serotonin slow inward current in identified snail neurones and have found that the intracellular injection of cyclic AMP, but not of cyclic GMP or 5'-AMP, evokes a slow inward current showing similar voltage dependence, inversion potential and ionic properties to the serotonin slow inward current. Phosphodiesterase inhibitors at low concentrations (1-20 microM) potentiate the serotonin slow inward current and at higher concentrations evoke by themselves an inward current, partially or totally occluding the serotonin and cyclic AMP currents. Finally, we have found that in homogenates of pooled identified snail neurones serotonin stimulates the adenylate cyclase, increasing its activity by 50-100%.

Effect of intracellular injection of cyclic amp on electrical characteristics of identified neurons in Helix pomatia

The effect of intracellular iontophoretic injection of cyclic AMP on electrical activity of neurons RPa1, RPa3, LPa2, LPa3, and LPl1 in the corresponding ganglia ofHelix pomatia was investigated. Injection of cyclic AMP into neuron LPl1 was found to cause the appearance of rhythmic activity (if the neuron was originally "silent"), an increase in the frequency of spike generation (if the neuron had rhythmic activity), and a decrease in amplitude of waves of membrane potential, in the duration of the interval between bursts, and in the number of action potentials in the burst (if the neuron demonstrated bursting activity). In the remaining "silent" neurons injection of cyclic AMP led to membrane depolarization. Injection of cyclic AMP into neurons whose membrane potential was clamped at the resting potential level evoked the development of an inward transmembrane current (cyclic AMP current), the rate of rise and duration of which increased proportionally to the size and duration of the injection. Theophylline in a concentration of 1 mM led to an increase in the amplitude and duration of the cyclic AMP current by about 50%. It is concluded that a change in the cyclic AMP concentration within the nerve cell may modify the ionic permeability of its membrane and, correspondingly, its electrical activity.

Presynaptic modulation of voltage-dependent Ca2+ current: mechanism for behavioral sensitization in Aplysia californica.

Behavioral sensitization of the gill-withdrawal reflex of Aplysia is the result of a prolonged increase in transmitter release from the presynaptic terminals of sensory neurons. Earlier work suggested that this presynaptic facilitation might be mediated by a serotonin-sensitive adenylate cyclase in the sensory neuron terminals. Here we present evidence that presynaptic facilitation results from a cyclic AMP-dependent increase in the calcium current that underlies action potentials in the sensory neurons. The action potentials of sensory neuron cell bodies have, in addition to a sodium current, a calcium current that is enhanced by blocking the opposing potassium current with tetraethylammonium. Under these conditions, the action potentials show a slowly repolarizing plateau that follows the Nernst potential for a calcium electrode and serves as a sensitive assay for changes in calcium current. Stimulation of the pathway that mediates sensitization, incubation with serotonin or phosphodiesterase inhibitors, or intracellular injection of cyclic AMP produces an increase in the calcium plateau in the presence of tetraethylammonium. In addition, both before and after sensitizing stimulation, the duration of the plateau potential parallels transmitter release as measured by the amplitude of monosynaptic excitatory postsynaptic potentials evoked in the motor neurons by intracellular stimulation of single sensory neurons. These results are consistent with the idea that presynaptic facilitation is caused by a cyclic AMP-mediated increase in a voltage-sensitive calcium current in sensory neuron presynaptic terminals. This synaptic action is novel in that it can produce little or no change in the resting potential, is of long duration, and exerts its influence directly on a conductance triggered by the action potential, rather than on non-voltage-sensitive conductances, as is typical of conventional synaptic actions.