Mechanical influence and cAMP injection evoke the same reaction of neuron ionic channels.

Intracellular cAMP injection and negative pressure in the patch-electrode increase the interburst closed time of the same potassium ionic channels in the snail neuron membrane. Sodium channels which were registered as change of background noise are activated both by cAMP injection and by negative pressure. These results are considered in connection with data about the unusual biochemistry of the neuron reaction to cAMP.     

The role of electro-mechanical and reaction-diffusion systems in the intra-neuron processing of information: effect of in-flow of calcium and intracellular calcium on cAMP activity

Influx of calcium ions cannot control a generatory potential induced by the intraneuronal system because calcium ions enter the cell during impulses. These impulses are the result of problem solving and must not influence directly the generatory potential. Therefore cAMP and not calcium controls the permeability of sodium and potassium channels from the inside of the neuron. However the calcium ions and membrane potential of mitochondria affect the impact of cAMP injections. An increase in the intracellular concentration of free Ca2+ induced by the injection of Ca-EGTA buffer with 5.10(-7) M free Ca2+, electric excitation, uncouplers of oxidative phosphorylation or arsenate leads to an increase of cAMP-dependent depolarization and the inward current. The injection of Ca-EGTA buffer with 10(-5) M free Ca2+ and drop in [Ca2+]in by EGTA as well as generation of impulses after cAMP injection decrease the cAMP effect. As rise in [Ca2+]in activates phosphodiesterase and uncouples oxidative phosphorylation, and vanadate in contrast to arsenate suppresses the cAMP effect, a hypothesis is advanced that activating effect of calcium on cAMP action is associated with neuron deenergization.     

A study of the change in conductivity of the neuronal membrane caused by a generator potential using a method of computer modeling

Injection of cAMP induces in snail neurons generator potential, which is related to an increase of sodium and decrease of potassium permeability of the neuron outer membrane. A model is proposed which takes into account cAMP diffusion inside the neuron from the injection place and interaction of these molecules with the intercellular system controlling permeability of the outer membrane. Resulting impulse generation induces calcium ions current through the outer membrane. The model also considers calcium diffusion toward cAMP and its effect on the rate of the enzyme work destroying cAMP. Agreement between the calculations of ionic current I(t) and the experiment permits determination of the model parameters and calculation of the observed change of time distribution of nerve impulses when calcium input is significant.     

Effect of high intracellular calcium ion concentration on the transmembrane current induced by iontophoretic injection of cAMP inHelix pomatia neurons

The effect of increasing the intracellular calcium ion concentration by various methods (iontophoretic injection into the cytoplasm, generation of a burst of action potentials, addition of uncouplers of oxidative phosphorylation to the external solution, causing release of calcium from mitochondria) on the inward current induced by injection of cAMP into the neuron (the cAMP current) was investigated on the neuron membrane ofHelix pomatia under voltage clamp conditions. In all cases an increase in the intracellular calcium ion concentration was found to lead to an increase in amplitude, and in many cases duration, of the cAMP current. It is suggested that membrane structures responsible for appearance of the cAMP current have two phosphorylation centers: cAMP-dependent and calcium-calmodulin-dependent. The possible role of this process in signal integration at the intraneuronal level is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 78–84, January–February, 1985.     

Effect of oxidative phosphorylation inhibitors and uncoupling agents on cAMP activity

Uncouplers of oxidative phosphorylation increased the speed of substrate oxidation and ATP hydrolysis and raised cAMP induced neuron membrane current. Different inhibitors decreased it. Both effects support the hypothesis that a signal of intracellular injected cAMP spreads to the neuron membrane as a mechanical signal. This signal propagated to the membrane along microtubules which according to this hypothesis serve as a sound generator with metabolic heat pumping.     

Intracellular injection of guanyl nucleotides alters the serotonin- induced increase in potassium conductance in Aplysia neuron R15

The effects of the adenylate cyclase inhibitor GDP beta S on the response of Aplysia neuron R15 to serotonin (5HT) were investigated. Previous studies have demonstrated that 5HT causes an increase in K+ conductance in R15 and that the response is mediated by cAMP. At concentrations in the micromolar range, GDP beta S inhibits the stimulation of adenylate cyclase by 5HT in particulate fractions from Aplysia ganglia. When micromolar concentrations of GDP beta S are injected into neuron R15, there is no effect on the resting membrane conductance, but the increase in K+ conductance normally elicited by 5HT is completely inhibited. Furthermore, the decrease in inward current normally elicited by dopamine (DA), which does not appear to involve cAMP, is not affected by micromolar concentrations of GDP beta S. In addition, application of 8-benzylthio cAMP to R15 can evoke an increase in K+ conductance even after the injection of GDP beta S, which indicates that events subsequent to the activation of adenylate cyclase are not inhibited by the GDP analogue. In contrast, when millimolar concentrations of GDP beta S are injected into R15, direct effects on membrane conductance are observed and the response of R15 to 5HT is enhanced. Although these effects of high concentrations of GDP beta S are only poorly understood, the results with micromolar concentrations are consistent with the hypothesis that stimulation of adenylate cyclase is necessary for the 5HT-induced increase in K+ conductance in neuron R15.     

Unusual biochemistry of changes in neuron membrane permeability evoked by cAMP

Influence of different metabolic poisons on cAMP-evoked neuron membrane permeability is investigated. Drugs preventing cAMP binding with R subunits of protein kinase decrease the cAMP-evoked current, but the inhibitor of the C subunit. H8, has no effect. The cAMP-dependent current is increased by uncouplers and decreased by inhibitors of glycolysis and oxidative phosphorylation. The mechanism of cAMP action on neuron permeability is discussed.     

The effect of various protein kinase inhibitors on the response of snail neurons to the intracellular injection of cAMP

Drugs preventing cAMP interaction with regulatory subunit of cAMP-dependent protein kinase, tolbutamide and db-cAMP injected into neurons of Helix lucorum decreased the cell response to cAMP, but H-8-a potent inhibitor of this enzyme catalytic subunit did not produce such effect. It is suggested that the neuron electric response to cAMP injection is not caused by protein phosphorylation.     

Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.     

The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation.

1. The involvement of protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity (BA) was evaluated in identified neurons of the snail. Euhadra peliomphala by examining the effect of various protein kinases and their inhibitors on the membrane properties induced by PTZ. 2. In neurons which normally exhibited spontaneous regular firing, PTZ elicited BA, the negative slope resistance (NSR) in the steady-state current (I)-voltage (V) relationship and a reduction of the delayed potassium current (IKD) in a dose-dependent manner. These were inhibited by the cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide. 3. Intracellular injection of catalytic subunit (CS) of cAMP-dependent protein kinase enhanced PTZ-induced NSR and reduction of IKD, as well as a conversion of the BA to a long-lasting depolarization of the membrane, whereas a saturating dose of the CS occluded PTZ action on the NSR and IKD. 4. Ca2+/calmodulin-dependent protein kinase II (CaMKII), when intracellularly injected during the depolarizing phase of PTZ-induced bursting cycle, changed to a prolonged hyperpolarization of the membrane. This kinase also restored the PTZ-suppressed IKD nearly to the pre-PTZ level. However, when intracellular injection of CaMKII and application of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin inhibitor, to the inside and outside of the neuron were simultaneously carried out, neither post-burst hyperpolarization nor restoration of the IKD was observed. 5. Intracellular injection of calmodulin, together with calcium chloride, had little effect on both the BA and reduction of IKD induced by PTZ. 6. Simultaneous application of 40 microM 1-(5-isoquinolinsulfonyl)-2-methylpiperazine, which selectively suppressed the phosphatidylserine-dependent protein phosphorylation in extracts from Euhadra ganglia, to both the inside and outside of the neuron, did not produce any significant change in the membrane properties induced by PTX. Intracellular injection of protein kinase C also brought about no effect. 7. These findings suggest that PTZ stimulates cAMP-dependent protein phosphorylation which, in turn, is involved in the development of NSR and reduction of IKD, leading to the depolarization of the membrane. In addition, we propose that the Ca2+ ions, increased during the depolarizing phase of the BA cycle, form a Ca2+/calmodulin complex and subsequent protein phosphorylation, coupled with the opening of potassium channels, leading to the membrane hyperpolarization.     

The cyclic GMP-induced inward current in neuron R14 of Aplysia californica: similarity to a FMRFamide-induced inward current

Injecting cGMP into Aplysia neuron R14 induced an inward current similar to one elicited by application of FMRFamide to the outside of that cell. In contrast, injection of cAMP into R14 caused a long-lasting outward current and conductance increase. Phosphodiesterase inhibitors increased the cGMP and FMRFamide-induced inward currents in R14. The cGMP-induced inward current is voltage dependent and is largely carried by Na+. It is also strongly and inversely dependent on both external [Ca2+] and [Cl-], although these ions are not significant current carriers. Changing external [K+] had no effect. Voltage and ion dependencies of the cGMP-induced inward current are similar to those of an inward current induced by FMRFamide. Thus cGMP may be a second messenger to FMRFamide in producing a slow inward current in R14. cGMP does not appear to be a second messenger to FMRFamide in most Aplysia neurons.     

A large contribution of a cyclic AMP-independent pathway to turtle olfactory transduction

Although multiple pathways are involved in the olfactory transduction mechanism, cAMP-dependent pathway has been considered to contribute mainly to the transduction. We examined the degree of contribution of cAMP-independent pathway to the turtle olfactory response by recording inward currents from isolated cells, nerve impulses from cilia and olfactory bulbar responses. The results obtained by the three recordings were essentially consistent with each other, but detail studies were carried out by recording the bulbar response to obtain quantitative data. Application of an odorant cocktail to the isolated olfactory neuron after injection of 1 mM cAMP from the patch pipette elicited a large inward current. Mean amplitude of inward currents evoked by the cocktail with 1 mM cAMP in the patch pipette was similar to that without cAMP in the pipette. Application of the cocktail after the response to 50 microM forskolin was adapted also induced a large inward current. Application of the odorant cocktail to the olfactory epithelium, after the response to 50 microM forskolin was adapted, brought about an appreciable increase in the impulse frequency. The bulbar response to forskolin alone reached a saturation level around 10 microM. After the response to 50 microM forskolin was adapted, 11 species of odorants were applied to the olfactory epithelium. The magnitudes of responses to the odorants after forskolin were 45-80% of those of the control responses. There was no essential difference in the degree of the suppression by forskolin between cAMP- and IP3- producing odorants classified in the rat, suggesting that certain part of the forskolin-suppressive component was brought about by nonspecific action of forskolin. Application of a membrane permeant cAMP analogue, cpt-cAMP elicited a large response, and 0.1 mM citralva after 3 mM cpt- cAMP elicited 51% of the control response which was close to the response to citralva after 50 microM forskolin. A membrane permeant cGMP analogue, db-cGMP elicited a small response and the response to 0.1 mM citralva was unaffected by db-cGMP. It was concluded that cAMP- independent (probably IP3-independent) pathway greatly contributes to the turtle olfactory transduction.     

The micronucleus test and erythropoiesis: effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2-O-dibutyladenosine-3',5'-cyclic monophosphate into adenosine 3',5'-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose-response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously. The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.     

Benzyl alcohol increases membrane fluidity and modulates cyclic AMP synthesis in intact renal epithelial cells

To evaluate a possible modulation by membrane fluidity of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of the neutral local anesthetic, benzyl alcohol, on membrane fluidity and on basal and stimulated intracellular cAMP content in intact MDCK cells. Benzyl alcohol induced a dose-dependent decrease of lipid order which was measured by steady-state fluorescence anisotropy using trimethylammonium-diphenylhexatriene and propionyl-diphenylhexatriene as fluorescent probes. Benzyl alcohol induced a 2-fold increase in basal cAMP content, likely as a consequence of increased prostaglandin synthesis since this effect was abolished by indomethacin. The effect of benzyl alcohol on stimulated cAMP synthesis depended on the nature of the ligand: 10 mM benzyl alcohol increased significantly the stimulatory effect of prostaglandin E2, glucagon and forskolin but not of vasopressin. At higher concentrations (40 mM), benzyl alcohol did not affect significantly the glucagon-stimulated cAMP content, while it inhibited significantly the prostaglandin E2-, forskolin- and vasopressin-stimulated cAMP synthesis. The 40 mM benzyl alcohol-induced inhibition was reversed by 1 mM Mn2+, which is known to block the inhibitory GTP-binding protein Ni. These results suggest that: (i) the various components of the adenylate cyclase-cAMP system and their coupling are affected differently by changes in membrane fluidity, which might reflect differences in their lipid environment, (ii) changes in membrane fluidity can modulate responses of renal tubular cells to hormones, and thus tubular functions.     

Phosphoproteins associated with the regulation of a specific potassium channel in the identified Aplysia neuron R15

The neurotransmitter serotonin (5HT) activates a specific K+ conductance in the identified Aplysia neuron R15. This response to 5HT has been shown previously to be mediated by cAMP and cAMP-dependent protein phosphorylation. We have measured protein phosphorylation within neuron R15 in vivo, following the intracellular injection of [gamma-32P]ATP, and have demonstrated that 5HT modulates the phosphorylation of a number of proteins in R15. The present study was undertaken to determine which of these phosphoproteins are closely associated with, and may be responsible for, the K+ conductance increase. Treatment of neuron R15 with a cAMP analog produces some but not all of the 5HT-induced phosphoprotein changes, indicating that some are not cAMP-dependent and thus can be dissociated from the cAMP-dependent K+ conductance increase. Similar results are obtained by intracellular injection of the adenylate cyclase inhibitor guanosine 5'-O-(2-thiodiphosphate), which completely blocks the 5HT-evoked K+ conductance increase but fails to block some of the 5HT-induced phosphorylation changes. Examination of the phosphoprotein pattern at short times after 5HT application has demonstrated that some of the phosphoprotein changes, but not others, are closely associated in time with the appearance of the physiological response. These and other pharmacological and kinetic experiments have allowed the identification of two phosphoproteins, of Mr = 29,000 and 70,000, which cannot be dissociated from the 5HT-induced K+ conductance increase whatever the experimental manipulation. Thus, one or both of these phosphoproteins may be involved in the regulation of the 5HT-sensitive K+ channel in neuron R15.     

Cationic membrane conductances induced by intracellularly elevated cAMP and Ca2+: measurements with ion-selective microelectrodes

Adenosine 3',5'-cyclic monophosphate (cAMP) and CaCl2 were injected by a fast and quantitative pressure injection technique into voltage-clamped, identified Helix neurons. Intracellular elevation of cAMP as well as of Ca2+ activated an inward current (IcAMP and IN). To identify the ionic fluxes during IcAMP and IN changes in [Na+]i, [K+]o, [H+]i, and [Cl-]i were measured with ion-selective microelectrodes (ISMs). Near resting potential, Na+ was the main carrier of IcAMP. K+, and less effectively Ca2+, could substitute for Na+ in carrying IcAMP. H+ and Cl- were excluded as current carriers for IcAMP by means of ISMs. Simultaneous to this action, cAMP decreased a K+ conductance. This decrease was associated with a reduction of the K+ efflux activated by long-lasting depolarizing voltage steps, as directly measured with ISMs located near the external membrane surface. The nearly compensatory increase and decrease of two membrane conductances in the same neuron left the cell input resistance unchanged despite the considerable depolarizing action of intracellularly elevated cAMP. IN was also of nonspecific nature. However, our findings indicate less selectivity for the Ca2+-activated nonspecific channels. Large cations such as choline, TEA, and Tris passed nearly as well as Na+ through the channels. Measurements with ISMs showed that [H+]i and [Cl-]i were unchanged during IN. IN was largest in bursting pacemaker neurons compared with other cells of similar size. It was found to be essential for the burst production in these cells. IcAMP, on the other hand, might be involved in the presynaptic facilitatory action of cAMP, which as yet was attributed solely to a reduction of a K+ conductance.     

cAMP/protein kinase A signalling pathway protects against neuronal apoptosis and is associated with modulation of Kv2.1 in cerebellar granule cells

Previously, we have reported that apoptosis of cerebellar granular neurons induced by incubation in 5 mm K(+) and serum-free medium (LK-S) was associated with an increase in the delayed rectifier K(+) current (I(K)). Here, we show that I(K) associated with apoptotic neurons is mainly encoded by a Kv2.1 subunit. Silencing Kv2.1 expression by small interfering RNA reduces I(K) and increases neuron viability. Forskolin is able to decrease the I(K) amplitude recording from neurons of both the LK-S and control group, and prevents apoptosis of granule cells that are induced by LK-S. Dibutyryl cAMP mimicks the effect of forskolin on the modulation of I(K) and, accordingly, the inhibitor of protein kinase A, H-89, aborts the neuron-protective effect induced by forskolin. Whereas the expression of Kv2.1 was silenced by Kv2.1 small interfering RNA, the inhibition of forskolin on the current amplitude was significantly reduced. Quantitative RT-PCR and whole-cell recording revealed that the expression of Kv2.1 was elevated in the apoptotic neurons, and forskolin significantly depressed the expression of Kv2.1. We conclude that the protection against apoptosis via the protein kinase A pathway is associated with a double modulation on I(K) channel properties and its expression of alpha-subunit that is mainly encoded by the Kv2.1 gene.     

Regulation of proliferation by the cholera toxin B subunit in FRTL-5 cells may involve a mechanism independent from the modulation of membrane receptor function

In quiescent rat thyroid (FRTL-5) cells, the B subunit of cholera toxin, which binds to cell surface ganglioside GM1 specifically, alone induced DNA synthesis and markedly enhanced that induced by insulin in serum-free medium. On the other hand, the B subunit inhibited DNA synthesis induced by thyrotropin (TSH). The B subunit did not activate adenylate cyclase and had no effect on the TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Moreover, the B subunit inhibited DNA synthesis induced by dibutyryl cAMP (Bt2cAMP) or phorbol-12-myristate-13-acetate (PMA). These data demonstrate that the B subunit has both stimulatory and inhibitory effects on DNA synthesis in FRTL-5 cells depending on the presence of other growth factors and that these effects on cell proliferation by the interaction of the B subunit, possibly with cell surface ganglioside GM1, may involve a mechanism independent from the modulation of membrane receptor function through interaction with growth factor receptor.     

Quantum molecular computer model of the neuron and a pathway to the union of the sciences

Cyclic nucleotide injection in neurons shows that cAMP controls a new type of membrane permeability. The neuron response to cAMP has a short delay, unusual bioenergetics and is blocked by drugs binding with the regulatory subunit of protein kinase. These data are interpreted in terms of the hypothesis that the controlling system of the living cell is a molecular (DNA, RNA, protein operators with complementary addresses), holographic (quick changeable lattice--cytoskeleton), quantum (each phonon examines whole lattice), hypersound (with wave length 100-10,000 A that does not destroy molecules) system with an inner point of view (molecular coding of questions and answers about quantum processing). Neither an electron, nor a macroscopic computer has an inner point of view.     

Change in neuronal membrane permeability exposed to adenosine-3',5'-cyclophosphate]

Ionic current induced by intracellular injection of cAMP was divided into constituent parts, and the dependence of these components on membrane potential and ionic composition of extracellular medium was demonstrated. The computation shows that practically all background neurone permeability for potassium ions is cAMP-dependent.