Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator

To investigate the dynamics of guanosine 3′,5′-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1–77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of ≈2 μM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr516 of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5–4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

Responses of the phototransduction cascade to dim light.

The biochemistry of visual excitation is kinetically explored by measuring the activity of the cGMP phosphodiesterase (PDE) at light levels that activate only a few tens of rhodopsin molecules per rod. At 23 degrees C and in the presence of ATP, the pulse of PDE activity lasts 4 s (full width at half maximum). Complementing the rod outer segments (ROS) with rhodopsin kinase (RK) and arrestin or its splice variant p44 does not significantly shorten the pulse. But when the ROS are washed, the duration of the signal doubles. Adding either arrestin or p44 back to washed ROS approximately restores the pulse width to its initial value, with p44 being 10 times more efficient than arrestin. This supports the idea that, in vivo, capping of phosphorylated R* is mostly done by p44. When myristoylated (14:0) recoverin is added to unwashed ROS, the pulse duration and amplitude increase by about 50% if the free calcium is 500 nM. This effect increases further if the calcium is raised to 1 microM. Whenever R* deactivation is changed--when RK is exogenously enriched or when ATP is omitted from the buffer--there is no impact on the rising slope of the PDE pulse but only on its amplitude and duration. We explain this effect as due to the unequal competition between transducin and RK for R*. The kinetic model issued from this idea fits the data well, and its prediction that enrichment with transducin should lengthen the PDE pulse is successfully validated.

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Origin and evolution of circular waves and spirals in Dictyostelium discoideum territories.

Randomly distributed Dictyostelium discoideum cells form cooperative territories by signaling to each other with cAMP. Cells initiate the process by sending out pulsatile signals, which propagate as waves. With time, circular and spiral patterns form. We show that by adding spatial and temporal noise to the levels of an important regulator of external cAMP levels, the cAMP phosphodiesterase inhibitor, we can explain the natural progression of the system from randomly firing cells to circular waves whose symmetries break to form double- and single- or multi-armed spirals. When phosphodiesterase inhibitor is increased with time, mimicking experimental data, the wavelength of the spirals shortens, and a proportion of them evolve into pairs of connected spirals. We compare these results to recent experiments, finding that the temporal and spatial correspondence between experiment and model is very close.

How photons start vision.

Recent studies have elucidated how the absorption of a photon in a rod or cone cell leads to the generation of the amplified neural signal that is transmitted to higher-order visual neurons. Photoexcited visual pigment activates the GTP-binding protein transducin, which in turn stimulates cGMP phosphodiesterase. This enzyme hydrolyzes cGMP, allowing cGMP-gated cationic channels in the surface membrane to close, hyperpolarize the cell, and modulate transmitter release at the synaptic terminal. The kinetics of reactions in the cGMP cascade limit the temporal resolution of the visual system as a whole, while statistical fluctuations in the reactions limit the reliability of detection of dim light. Much interest now focuses on the processes that terminate the light response and dynamically regulate amplification in the cascade, causing the single photon response to be reproducible and allowing the cell to adapt in background light. A light-induced fall in the internal free Ca2+ concentration coordinates negative feedback control of amplification. The fall in Ca2+ stimulates resynthesis of cGMP, antagonizes rhodopsin's catalytic activity, and increases the affinity of the light-regulated cationic channel for cGMP. We are using physiological methods to study the molecular mechanisms that terminate the flash response and mediate adaptation. One approach is to observe transduction in truncated, dialyzed photoreceptor cells whose internal Ca2+ and nucleotide concentrations are under experimental control and to which exogenous proteins can be added. Another approach is to observe transduction in transgenic mouse rods in which specific proteins within the cascade are altered or deleted.

Gain and kinetics of activation in the G-protein cascade of phototransduction.

The guanine nucleotide binding protein (G protein) cascade underlying phototransduction is one of the best understood of all signaling pathways. The diffusional interactions of the proteins underlying the cascade have been analyzed, both at a macroscopic level and also in terms of the stochastic nature of the molecular contacts. In response to a single activated rhodopsin (R*) formed as a result of a single photon hit, it can be shown that molecules of the G-protein transducin will be activated approximately linearly with time. This, in turn, will cause the number of activated molecules of the effector protein (the phosphodiesterase) also to increase linearly with time. These kinetics of protein activation provide an accurate description of the time course of the rising phase of the photoreceptor's electrical response over a wide range of flash intensities. Recent estimates indicate that at room temperature each R* triggers activation of the phosphodiesterase at a rate of 1000-2000 subunits.s-1. Now that a quantitative description of the activation steps in transduction has been obtained, perhaps the greatest challenge for the future is to provide a comprehensive description of the shutoff reactions, so that a complete account of the photoreceptor's response to light can be achieved.

The biology of vision of Drosophila.

Phototransduction systems in vertebrates and invertebrates share a great deal of similarity in overall strategy but differ significantly in the underlying molecular machinery. Both are rhodopsin-based G protein-coupled signaling cascades displaying exquisite sensitivity and broad dynamic range. However, light activation of vertebrate photoreceptors leads to activation of a cGMP-phosphodiesterase effector and the generation of a hyperpolarizing response. In contrast, activation of invertebrate photoreceptors, like Drosophila, leads to stimulation of phospholipase C and the generation of a depolarizing receptor potential. The comparative study of these two systems of phototransduction offers the opportunity to understand how similar biological problems may be solved by different molecular mechanisms of signal transduction. The study of this process in Drosophila, a system ideally suited to genetic and molecular manipulation, allows us to dissect the function and regulation of such a complex signaling cascade in its normal cellular environment. In this manuscript I review some of our recent findings and the strategies used to dissect this process.

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.