Gated conductances in native and reconstituted membranes from frog olfactory cilia.

Although cAMP is well established as a second messenger for olfactory transduction in vertebrates, the role of inositol 1,4,5-trisphosphate (IP3) in this process remains controversial. We addressed this issue by comparing currents evoked by cAMP and IP3 in native and reconstituted membranes from olfactory cilia. We detected only a cyclic nucleotide-gated conductance in the native membrane but both cyclic nucleotide-gated and IP3-gated conductances in the reconstituted membrane. The magnitudes of the cyclic nucleotide- and IP3-gated conductances were not correlated with each other in reconstituted membranes, suggesting that cyclic nucleotide- and IP3-gated channels originate in different cellular compartments.     

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Fifty years ago, cyclic AMP was discovered as a second messenger of hormone action, heralding the age of signal transduction. Many cellular processes were found to be regulated by cAMP and the related cyclic GMP. Cyclic nucleotides function by binding to and activating their effectors - protein kinase A, protein kinase G, cyclic-nucleotide-regulated ion channels and the guanine nucleotide-exchange factor Epac. Recent structural insights have now made it possible to propose a general structural mechanism for how cyclic nucleotides regulate these proteins.     

Variable acceleration influences cyclic AMP levels in Paramecium biaurelia

Paramecium is used as a model system to analyse the gravity signal transduction pathway, that leads to gravitaxis and gravikinesis. In order to prove whether gravistimulation is coupled with second messenger production (cyclic AMP: hyperpolarization, cyclic GMP: depolarization) Paramecium was fixated under variable accelerations (1 x g, 9 x g and 10(-4) x g) on a centrifuge and during a sounding rocket flight (TEXUS 39). The analysis of cAMP and cGMP levels revealed an acceleration-dependent change in cAMP, while cGMP-levels showed gravity-independent variations. Hypergravity did not only induce an amplification of gravitaxis and gravikinesis, but also an increase in cAMP compared to the 1 x g-data. We conclude that the increased pressure of the cytoplasm on the lower membrane of upward swimming cells enhance the number of open K+(-)channels, thus causing hyperpolarization and change in cAMP concentration. Consequently, transition to microgravity declines gravitaxis and gravikinesis, and decreases cAMP concentration due to the loss of pressure on the cell membrane.     

The cyclic nucleotide gated channel of olfactory receptor neurons

Olfactory transduction proceeds through a G-protein coupled cascade that produces the ubiquitous second messenger cyclic AMP. The cyclic AMP causes a chance in membrane potential by acting directly on an ion channel that allows cations to flow into the cell. This ion channel is one of a new family of ion channels that are activated by intracellular cyclic nucleotides. However, even though they are activated by binding a ligand their amino acid structure shows that they share a common ancestry with voltage activated channels, especially voltage dependent Ca²⁺ channels. In olfactory neurons these channels perform a critical role in the transduction of chemical information in the environment into changes in membrane electrical properties that are transmitted to higher order processing centers in the brain.     

Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation

cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.     

Sea urchin sperm cation-selective channels directly modulated by cAMP

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm-egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation-selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.     

The second messenger cascade in olfactory receptor neurons.

The molecular cloning of components involved in the cAMP second messenger cascade has allowed their biochemical characterization and revealed properties that are important for their role in sensory transduction. Recent evidence suggests inositol 1,4,5-trisphosphate functions as an additional second messenger in olfactory signalling. The interaction of these two pathways may contribute to the sensitivity of the olfactory system.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

Epinephrine-induced pigment aggregation in goldfish melanophoroma cells: apparent involvement of an unknown second messenger

Using a goldfish-derived melanized cell line, we attempted to determine the identity of the signal transduction system/second messenger for epinephrine-induced aggregation of melanosomes in a goldfish cell line. The results show that the second messenger is unknown. It is not 1) influx of extracellular calcium, 2) release of intracellular stored calcium via the phosphoinositide pathway, 3) cGMP, or 4) decrease of cAMP. These results suggest that there is an unknown second messenger for this activity of epinephrine.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Neural cell adhesion molecules influence second messenger systems

We have investigated the influence of the neural cell adhesion molecules L1 and N-CAM on second messenger systems using a PC12 rat pheochromocytoma cell line as a model and triggering cell surface receptors by specific antibody binding. Antibodies directed against L1 and N-CAM, but not against other cell surface components, reduce intracellular levels of the inositol phosphates IP2 and IP3, while intracellular levels of cAMP are unaffected. Antibodies against L1 and N-CAM also reduce intracellular pH and increase intracellular Ca2+ by opening Ca2+ channels in a pertussis toxin-inhibitable manner, suggesting the involvement of a G protein in the signal transduction process. Cross-linking of the adhesion molecules on the surface membrane is not required for the effects to occur. Furthermore, adhesion of single PC12 cells to each other elicits effects on intracellular pH and Ca2+ similar to those seen after application, underscoring the physiological significance of the observed changes.     

Role of soluble adenylyl cyclase in the heart

This review discusses the potential place of soluble adenylyl cyclase (sAC) in the framework of signaling in the cardiovascular system. cAMP has been studied as a critical and pleiotropic second messenger in cardiomyocytes, endothelial cells, and smooth muscle vascular cells for many years. It is involved in the transduction of signaling by catecholamines, prostaglandins, adenosine, and glucagon, just to name a few. These hormones can act via cAMP by binding to a G protein-coupled receptor on the plasma membrane with subsequent activation of a heterotrimeric G protein and its downstream effector, transmembrane adenylyl cyclase. This has long been the canonical standard for cAMP production in a cell. However, the relatively recent discovery of a unique source of cAMP, sAC, creates the potential for a shift in this signaling paradigm. In fact, sAC has been shown to play a role in apoptosis in coronary endothelial cells and cardiomyocytes. Additionally, it links nutrient utilization with ATP production in the liver and brain, which suggests one of many potential roles for sAC in cardiac function. The possibility of producing cAMP from a source distal to the plasma membrane provides a critical new building block for reconstructing the cellular signaling infrastructure.     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

Selective permeability of different connexin channels to the second messenger cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.     

Triggering of T-lymphocytes via either T3-Ti or T11 surface structures opens a voltage-insensitive plasma membrane calcium-permeable channel: requirement for interleukin-2 gene function

Stimulation of human T-lymphocytes via either the surface T3-Ti antigen-major histocompatibility complex receptor complex or the T11 molecule results in clonal proliferation through a calcium-dependent mechanism. To investigate this signal transduction, plasma membrane calcium-permeable channels were characterized in T-lymphocytes by means of whole cell or single channel patch-clamp recordings. Stimulation of T-lymphocytes via either structure results in opening of an identical set of voltage-insensitive plasma membrane Ca2+-permeable channels through the action of a diffusible second messenger. Previous work with excised inside-out patches suggests that inositol 1,4,5-trisphosphate is the activating second messenger of the voltage-insensitive T-cell Ca2+-permeable channel. Since there is a significant increase in phosphoinositide turnover after stimulation via either the T3-Ti or T11 pathway, it is suggested that triggering of either structure opens a common set of channels through this mechanism. Furthermore, currents flowing through Ca2+-permeable channels are apparently autoregulated, as inward conductance is abolished by elevation of Ca2+ concentration in the bathing solution. In particular, the steady-state rise in interleukin-2 (T-cell growth factor) mRNA is dependent on the rise of [Ca2+]i resulting from ion movement across this channel.     

cAMP: novel concepts in compartmentalised signalling

Cyclic adenosine 3,'5'-monophosphate (cAMP) is the archetypal second messenger produced at the membrane by adenylyl cyclase following activation of many different G protein-coupled receptor (GPCR) types. Although discovered over fifty years ago, the notion that cAMP responses were compartmentalised was born in the 1980s. Since then, modern molecular techniques have facilitated visualisation of cellular cAMP dynamics in real time and helped us to understand how a single, ubiquitous second messenger can direct receptor-specific functions in cells. The aim of this review is to highlight emerging ideas in the cAMP field that are currently developing the concept of compartmentalised cAMP signalling systems.     

Anchored cAMP signaling: onward and upward - a short history of compartmentalized cAMP signal transduction

Intracellular signal transduction pathways require a high degree of spatial and temporal resolution in order to deliver the appropriate outputs. Specific signaling mediated by the ubiquitous second messenger cAMP and its effector, the cAMP-dependent protein kinase (PKA), is governed by the spatial organization of different pathway components by A-kinase anchoring proteins (AKAPs). This review discusses the history and future of anchored cAMP signaling pathways.     

Anchored cAMP signaling: Onward and upward – A short history of compartmentalized cAMP signal transduction

Intracellular signal transduction pathways require a high degree of spatial and temporal resolution in order to deliver the appropriate outputs. Specific signaling mediated by the ubiquitous second messenger cAMP and its effector, the cAMP-dependent protein kinase (PKA), is governed by the spatial organization of different pathway components by A-kinase anchoring proteins (AKAPs). This review discusses the history and future of anchored cAMP signaling pathways.