Four cases of direct ion channel gating by cyclic nucleotides

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 µM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10–15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked byl-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1–40 µM. The channel is poorly cationic selective. A K⁺ channel directly and specifically activated by cAMP is found inDrosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 µM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10–20-fold larger in the mutantdunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Requirement for the action of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus

The role of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus L. was investigated. The seeds readily germinated in water and darkness at 24°C. Application of ethylene or of its precursor I-aminocyclopropane-I-carboxylic acid (ACC) slightly increased the rate of germination. Both compounds effectively antagonized osmotic inhibition by polyethyleneglycol. Application of aminoethoxyvinylglycine (AVG) reduced ethylene production by 90% but did not inhibit germination. However, germination was inhibited by 2,5-norbornadiene, a competitive inhibitor of ethylene action. This inhibition was counteracted by ethylene, ethephon or ACC and enforced by AVG. It is concluded that the action of endogenous ethylene is an indispensable factor during germination of non-dormant seeds of A. caudatus. Ethylene action is required from the start of imbibition on. In water, low levels of endogenous ethylene are sufficient for this action. PEG increased the ethylene requirement considerably.     

Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory - An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose-bis-phosphate-carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin-isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.