The enzymatic neuron as a reaction-diffusion network of cyclic nucleotides

Recent evidence suggests that the cyclic nucleotides play a central role in the intracellular processing of neural signals. The dynamics of this system may be seen as a realization of the enzymatic neuron model. Enzymatic neurons are formal neurons which map binary afferent signals into patterns of excitation across an abstract membrane. The distribution of enzyme-like elements called excitases enables a set of local threshold functions to determine the firing activity of the neuron. This paper analyzes the basic properties of enzymatic neurons in a simple continuous-time framework, and shows how they may be presented as reaction-diffusion networks which model the cyclic nucleotide system. We present the results of computer simulations of this neuron and discuss its implications for selectional learning and its relation to conventional two-factor systems. One fundamental property of the reaction-diffusion neuron is its so-called “double-dynamics” property; examination of this property and its contribution to the computing power of the neuron provides some insight into the obscure relation between microscopic and macroscopic models of computation.     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Role of cyclic nucleotides in modulating ovarian hCG action.

The role of cyclic nucleotides in mediating hormonally responsive adenylate cyclase and cAMP-dependent protein kinase was examined in vivo and in vitro when pseudopregnant rats were injected with hCG. Intracellular ovarian levels of cAMP increased, as expected, but no change in cGMP concentrations was observed. However, both cGMP and cAMP activated ovarian CDPK holoenzyme in vitro but cGMP had a lower affinity. The subunits of hCG were without effect. Even though cGMP and cAMP dissociate partially purified ovarian CDPK holoenzyme in vitro, the receptor sites of the regulatory subunit of CDPK would appear to be relatively specific for cAMP. Moreover, cGMP probably does not mediate hCG action in vivo.     

Light-dependent K(+) channels in the mollusc Onchidium simple photoreceptors are opened by cGMP

Light-dependent K(+) channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268-272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K(+) channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K(+) and 200 mM Mg(2+) to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K(+) concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants tau(o1), tau(o2) and tau(c1), tau(c2), respectively, similar to those of the light-dependent channel. In both the channels, tau(o1) and tau(o2) in ms ranges were similar to each other, although tau(c2) over tens of millisecond ranges was different. tau(o1), tau(o2), and the mean open time tau(o) were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of tau(o2) or tau(o). In both, the reversal potentials using 200- and 450-mM K(+)-filled pipettes were close to the K(+) equilibrium potentials, suggesting that both the channels are primarily K(+) selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K(+) pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K(+) channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.     

Reduction of a model for an Onchidium pacemaker neuron

The eight-variable model for the giant neuron localized in the esophageal ganglia of the marine pulmonate mollusk Onchidium verruculatum is reduced to four-and-three-dimensional systems by regrouping variables with similar time scales. These reduced models replicate the complex behavior including beating, periodic bursting and aperiodic bursting displayed by the original full model when the parameter I _ext representing the intensity of the constant DC current stimulation is varied across a wide range. The complex behavior of the full model arises from the interaction of fast and slow dynamics, and depends on the time scale C _s of the slow dynamics. The four-variable reduced model is constructed independently from the parameter C _s so that it reproduces the two-dimensional bifurcation structure of the full model for the two parameters I _ext and C _s . The three-variable reduced model is derived for a specific value of C _s . The parameters of this model are tuned so that its one-parameter bifurcation diagram for I _ext closely matches that of the full model. Correspondence between bifurcation structures ensures that both reduced models reproduce the various discharge patterns of the full model. Similarity between the full and reduced models is also confirmed by comparing mean firing frequencies and membrane potential waveforms in various regimes. The reduction exposes the factors essential for reproducing the dynamics of the full model; indeed, it shows that the eight variables representing the membrane potential and seven gating variables of six ionic currents in the full model account, in fact, for three basic processes responsible for excitability, post-discharge refractoriness and slow membrane modulation. Received: 7 May 1996 / Accepted 4 December 1997     

Role and place of cyclic nucleotides in the neuroendocrine regulation of cells and tissues

Cyclic adenosine monophosphate and cyclic guanosine monophosphate occupy one of the central places in the neuro-endocrine regulation in the highest organisms. These nucleotides mediate the effect of most hormones and neurotransmitters: peptide, polypeptide and proteinaceous, catecholamine, histamine, many prostaglandins, acetylcholine (in the case of muscarinic cholinergic receptor activation), endorphins and enkephalins. They regulate also synthesis and secretion of many hormones and neurotransmitters. Cyclic nucleotides play a key role in the functioning of organs of sense (vision, smelling and taste). Under the control of cyclic nucleotides there are main metabolic processes: glycogenolysis and lipolysis. These nucleotides have been shown to participate in the genome expression, in growth, differentiation and proliferation of cells. Accelerating or decelerating the ionic transport across the biological membranes cyclic nucleotides influence the cell bioelectric activity as well its contractility. They may also change the properties of contractile and cytoskeleton proteins, thus interfering in the processes of mobility and shape formation of cells.     

Saturation of anti cyclic nucleotides antibodies by endogenous cyclic nucleotides.

Rabbits immunized against cyclic AMP or cyclic GMP produce antibodies which are fully saturated by their respective endogenous cyclic nucleotides. This was proved a) in comparing radioimmunological measurements of cyclic nucleotides in antiserum and the binding site concentration determined by equilibrium dialysis, b) in showing the ineffectiveness of serum phosphodiesterase to hydrolyze the cyclic AMP present in the anti-cyclic AMP antiserum. Immunological and radioimmunological implications of this phenomenon are discussed.     

Surface charges on the outer side of mollusc neuron membrane

Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK _Ca=90±10,K _Sr=60±10,K _Ba=25±5 andK _Mg=16±5m ^–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK _Ca=67±10,K _Sr=20±5 andK _Ba=19±5m ^–1 at pH=7.0. The same groups bind H⁺ ions with apparent pK=6.2±0.2 that corresponds toK _H=1.6×10⁶ m ^–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm² (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm² (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.     

Role of cyclic nucleotides in the regulation of mitochondrial calcium uptake and efflux kinetics.

The effects of cyclic AMP and/or cyclic GMP upon various mitochondrial parameters were investigated. It was found that these nucleotides were unable to promote either inhibition of Ca²⁺ uptake and/or efflux or phosphorylation of ADP. These results are in contrast with those of other investigators, and suggest that cyclic nucleotides do not chemically mediate mitochondrial activity.     

Phosphodiesterase of cyclic nucleotides

Problems are considered on form multiplicity, purification and molecular weight of cyclic nucleotides phosphodiesterase. A supposition is made that the molecular weight of the catalytic subunit of the enzyme for most studied objects is about 60000. The catalytic subunit may form di- and trimers and be associated with regulatory proteins of different type. The problem of phosphodiesterase regulation is analyzed on the basis of potentialities of the equilibrium shift between the protein subunits of the enzyme; the role of cyclic nucleotides as well as of triphosphonucleotides are shown to influence the regulation of the enzymic activity. In some cases the mechanism of changes in the activity of phosphodiesterase bound with the receptor is shown to be similar to that for adenylate cyclase. In particular, the role of GTP and one of the protein subunits of phosphodiesterase in this process is stated.     

ACTH,cyclic nucleotides,and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated³²P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH_1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10^–10 to 10^–4 M. In a number of protein bands, a biphasic effect of ACTH_1–24 was observed: in concentrations of 10^–4–10^–5 M, a reduced amount of³²P was found; in concentrations of 10^–6–10^–7 M, hardly any effect could be detected, whereas consistently at concentrations around 10^–8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.     

Effect of cyclic nucleotides and phosphodiesterase inhibition on morphine tolerance and physical dependence.

The effects of cyclic nucleotides and theophylline were assessed in mice rendered tolerant to and physically dependent on morphine by the pellet implantation procedure. Tolerance was quantified by the increase in amount of morphine to produce analgesia and dependence by the decrease in amount of naloxone to precipitate withdrawal jumping. By these criteria, pretreatment with a single intravenous injection of cyclic 3′, 5′-adenosine monophosphate (cAMP) was found to enhance markedly tolerance and dependence development. Repeated injections of theophylline were also affective. Cycloheximide and beta-adrenergic blockers prevented the accelerating effect of cAMP and with more frequent administration also decreased the development of tolerance and dependence. It is concluded that cAMP may have a role in morphine tolerance and dependence development.     

Regulation of cyclic nucleotides in retinal photoreceptors

Summary The distribution of cyclases in retinal photoreceptors of dark- and light-adapted brook trout was studied by means of a cytochemical method (lead precipitation). It confirms earlier reports that retinal photoreceptors contain high levels of cyclic nucleotides, and that cAMP predominates in cones and cGMP in rods. There is an apparent difference in the level of the cyclases with the adaptive states. In addition, the catalytic unit of cyclase is interlamellar in cones. In rods, adenylate cyclase is intradiscal, while the location of guanylate cyclase varies with the adaptive state. The variation of cyclase with adaptation indicates that this enzyme has a role in the process of visual transduction.