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.     

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.     

Intraneuronal information processing. Delay in the changes in membrane potential after administration of cyclic nucleotides

Depolarization of the neuron membrane induced by cyclic adenosine monophosphate (cAMP) was shown both by ionophoresis and by injection with pressure. Swelling of the neuron during the injection of various substances with pressure causes membrane depolarization which is similar to that induced by cAMP. When applying pressure the cAMP effect can be distinguished by introducing small volumes of concentrated solutions. Similarity between the effects of cAMP and mechanical stimulation of the neuron suggests that in both cases the effect involves action of the electromechanical system consisting of microskeleton and micromuscles which regulate permeability of molecular channels. The delay of the effect after the moment of cAMP and cGMP introduction is small, which enables a conclusion concerning their direct interaction with the cytoskeleton.     

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.     

Cyclic AMP enhances calcium-dependent potassium current inAplysia neurons

The effect on the Ca-dependent potassium current, IK(Ca), of procedures that increase intracellular cAMP levels was studied in Aplysia neurons using three different pharmacological approaches. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase in IK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increased IK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. Application of serotonin plus phosphodiesterase inhibitor caused an increase in IK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease in IK(Ca). It is proposed that elevation of cyclic AMP mediates an increased sensitivity of the IK(Ca) channel to Ca ions.     

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.     

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.     

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.     

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.     

Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.     

NO-Evoked macrophage apoptosis is attenuated by cAMP-induced gene expression

BACKGROUND: Previous work has suggested that an increase in expression of cyclooxygenase-2, concomitant formation of E-type prostanoids, and in turn intracellular cAMP conveys macrophage resistance against apoptosis. MATERIALS AND METHODS: We analyzed the effects of lipophilic cAMP analogs on nitric oxide (NO)-induced apoptosis in RAW 264.7 macrophages and human primary monocyte-derived macrophages. Parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarization (DeltaPsi), analyzed using DiOC(6)(3). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c, and expression of the antiapoptotic protein Bcl-X(L). A cAMP response-element decoy approach confirmed cAMP-dependent gene induction. RESULTS: We verified resistance of murine and human macrophages against NO donors such as S-nitrosoglutathione or spermine-NO by pre-exposing cells to lipophilic cAMP analogs or by pretreatment with lipopolysaccaride, interferon-gamma, and N(G)-nitroarginine-methylester for 15 hr. Cellular prestimulation decreased NO-evoked apoptosis, as apoptotic parameters were basically absent. Macrophage protection was not achieved during a short period of preexposure, i.e., 1 hr. To verify gene induction as the underlying protective principle, we treated RAW cells with oligonucleotides containing a cAMP-responsive element in order to scavenge cAMP response element-binding protein prior to its promoter-activating ability. Decoy oligonucleotides, but not an unrelated control oligonucleotide, weakened cAMP-evoked protection and re-established a p53 response following NO addition. CONCLUSION: Gene induction by cAMP protects macrophages against apoptosis that occurs as a result of excessive NO formation. Decreasing programmed cell death of macrophages may perpetuate inflammatory conditions in humans when macrophages become activated in close association with innate immune responses.     

Rap1 activation plays a regulatory role in pancreatic amylase secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.     

Potential-dependent membrane sodium pump current in snail giant neurons

The effect of the membrane potential on the pump current evoked by iontophoretic injection of sodium into the neuron and the effect of the intracellular sodium ion concentration on the potential dependence of the pump current were investigated by the voltage clamp method in isolated and semi-isolated neurons ofHelix pomatia andHelix italiana. The pump current was shown to change its direction in the presence of marked hyperpolarization of the membrane (by more than −80 to −120 mV). An increase in the intracellular sodium ion concentration following injection of excess ions into the neuron increases the potential dependence of the pump current. A possible connection between passive potassium permeability and the activity of the enzymic transport mechanism for the elimination of sodium from the cell is postulated.     

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.     

Second messengers of octopamine receptors in the snail Lymnaea

We describe octopamine responses of 3 large buccal neurons of Lymnaea and test the hypothesis that these are cAMP-dependent. The B1 neuron is excited by octopamine and the depolarisation is significantly enlarged (P < 0.05) by application of the blocker of cAMP breakdown, 3-isobutyl-1-methylxanthine (IBMX). The B1 neuron is also depolarised by forskolin, an activator of adenylyl cyclase. The B2 and B3 neurons are inhibited by octopamine, and the response is not affected by IBMX. Both cells are excited by forskolin. We conclude that the B1 neuron response to octopamine is likely to be mediated by cAMP, while the B2 and B3 responses are cAMP-independent.     

Prostaglandin E2, adenosine 3':5'-cyclic monophosphate and changes in endometrial vascular permeability in rat uteri sensitized for the decidual cell reaction

The possibility that prostaglandin E2 (PGE2) increases endometrial vascular permeability and initiates decidualization in sensitized rat uteri by stimulation of adenosine 3':5'-cyclic monophosphate (cAMP) synthesis was investigated. Immature rats, pretreated so that they were sensitized for the decidual cell reaction, were used. Following the unilateral intrauterine injection of 50 microliters phosphate-buffered saline containing gelatin (PBS-G), a deciduogenic stimulus, uterine concentrations of both PGE and cAMP were elevated as early as 1 min after the intrauterine treatment. To determine if uterine stimuli which increase endometrial vascular permeability also increase uterine cAMP concentrations, rats, treated with or without indomethacin, an inhibitor of PG synthesis, received unilateral intrauterine injections of 50 microliters PBS-G with and without 10 micrograms PGE2 and were killed 15 min later. Uterine cAMP concentrations were elevated in all injected horns except in those of indomethacin-treated rats receiving PBS-G intraluminally, thus paralleling the expected changes in endometrial vascular permeability. As indicated by radioactivity levels in the stimulated horn 15 min after the i.v. injection of 125I-labeled bovine serum albumin, the intrauterine injection of dibutyryl cAMP, with or without theophylline, did not increase endometrial vascular permeability in indomethacin-treated animals. In contrast, cholera toxin, an activator of adenylate cyclase activity, markedly elevated permeability and induced decidualization. Except for the lack of a permeability response to the cAMP analogue, these data are consistent with the hypothesis that the effect of PGE2 on endometrial vascular permeability is mediated by cAMP.     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

Contribution of cyclic-nucleotide-gated channels to the resting conductance of olfactory receptor neurons

The basal conductance of unstimulated frog olfactory receptor neurons was investigated using whole-cell and perforated-patch recording. The input conductance, measured between -80 mV and -60 mV, averaged 0.25 nS in physiological saline. Studies were conducted to determine whether part of the input conductance is due to gating of neuronal cyclic-nucleotide-gated (CNG) channels. In support of this idea, the neuronal resting conductance was reduced by each of five treatments that reduce current through CNG channels: external application of divalent cations or amiloride; treatment with either of two adenylate cyclase inhibitors; and application of AMP-PNP, a competitive substrate for adenylate cyclase. The current blocked by divalent cations or by a cyclase inhibitor reversed near 0 mV, as expected for a CNG current. Under physiological conditions, gating of CNG channels contributes approximately 0.06 nS to the resting neuronal conductance. This implies a resting cAMP concentration of 0.1-0.3 micro M. A theoretical model suggests that a neuron containing 0.1-0.3 micro M cAMP is poised to give the largest possible depolarization in response to a very small olfactory stimulus. Although having CNG channels open at rest decreases the voltage change resulting from a given receptor current, it more substantially increases the receptor current resulting from a given increase in [cAMP].     

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.