RESUMEN
SOCE (store-operated Ca2+ entry) is mediated via specific plasma membrane channels in response to ER (endoplasmic reticulum) Ca2+ store depletion. This route of Ca2+ entry is central to the dynamic interplay between Ca2+ and cAMP signalling in regulating the activity of Ca2+-sensitive adenylate cyclase isoforms (AC1, AC5, AC6 and AC8). Two proteins have been identified as key components of SOCE: STIM1 (stromal interaction molecule 1), which senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion, where it then activates Orai1, the pore-forming component of the CRAC (Ca2+ release-activated Ca2+) channel. Previous studies reported that co-expression of STIM1 and Orai1 in HEK-293 (human embryonic kidney 293) cells enhances Ca2+-stimulated AC8 activity and that AC8 and Orai1 directly interact to enhance this regulation. Nonetheless, the additional involvement of TRPC (transient receptor potential canonical) channels in SOCE has also been proposed. In the present study, we evaluate the contribution of TRPC1 to SOCE-mediated regulation of Ca2+-sensitive ACs in HEK-293 cells stably expressing AC8 (HEK-AC8) and HSG (human submandibular gland) cells expressing an endogenous Ca2+-inhibited AC6. We demonstrate a role for TRPC1 as an integral component of SOCE, alongside STIM1 and Orai1, in regulating Ca2+ fluxes within AC microdomains and influencing cAMP production.
Asunto(s)
Adenilil Ciclasas/fisiología , Señalización del Calcio/fisiología , Canales Catiónicos TRPC/fisiología , Animales , Calcio/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratas , Glándula Submandibular/metabolismoRESUMEN
Adenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca(2+)-sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca(2+) entry (Willoughby et al., 2010). Here, we reveal that AKAP79 recruits cAMP-dependent protein kinase (PKA) to mediate the regulatory effects of AKAP79 on AC8 activity. Modulation by PKA is a novel means of AC8 regulation, which may modulate or apply negative feedback to the stimulation of AC8 by Ca(2+) entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56δ subunits that associate with the N-terminus of AC8. By site-directed mutagenesis we identify Ser-112 as an essential residue for direct PKA phosphorylation of AC8 (Ser-112 lies within the N-terminus of AC8, close to the site of AKAP79 association). During a series of experimentally imposed Ca(2+) oscillations, AKAP79-targeted PKA reduced the on-rate of cAMP production in wild-type but not non-phosphorylatable mutants of AC8, which suggests that the protein-protein interaction may provide a feedback mechanism to dampen the downstream consequences of AC8 activation evoked by bursts of Ca(2+) activity. This fine-tuning of Ca(2+)-dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca(2+) and cAMP, such as pulsatile hormone secretion and memory formation.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Western Blotting , Línea Celular , Humanos , Inmunoprecipitación , FosforilaciónRESUMEN
PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Lipoilación , Microdominios de Membrana/metabolismo , Animales , Línea Celular , Centrifugación por Gradiente de Densidad , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cisteína/metabolismo , Difusión/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Inositol/metabolismo , Lipoilación/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Octoxinol/farmacología , Palmitatos/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Receptores Adrenérgicos beta/metabolismo , Solubilidad/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sistemas de Mensajero Secundario/fisiología , Proteínas de Anclaje a la Quinasa A/genética , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Enfermedad Crónica , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Ratas , Ratas Endogámicas WKY , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Microdominios de Membrana/metabolismo , Sondas Moleculares/metabolismo , Isoformas de Proteínas/metabolismo , Adenilil Ciclasas/genética , Señalización del Calcio , Línea Celular , AMP Cíclico/metabolismo , Citosol , Humanos , Sondas Moleculares/genética , Unión Proteica , Ingeniería de Proteínas , Isoformas de Proteínas/genética , Transporte de Proteínas/genéticaRESUMEN
Cross-talk between cAMP and Ca2+ signalling pathways plays a critical role in cellular homoeostasis. Several AC (adenylate cyclase) isoforms, catalysing the production of cAMP from ATP, display sensitivity to submicromolar changes in intracellular Ca2+ and, as a consequence, are key sites for Ca2+ and cAMP interplay. Interestingly, these Ca2+-regulated ACs are not equally responsive to equivalent Ca2+ rises within the cell, but display a remarkable selectivity for regulation by SOCE (store-operated Ca2+ entry). Over the years, considerable efforts at investigating this phenomenon have provided indirect evidence of an intimate association between Ca2+-sensitive AC isoforms and sites of SOCE. Now, recent identification of the molecular components of SOCE [namely STIM1 (stromal interaction molecule 1) and Orai1], coupled with significant advances in the generation of high-resolution targeted biosensors for Ca2+ and cAMP, have provided the first detailed insight into the organization of the cellular microdomains associated with Ca2+-regulated ACs. In the present review, I summarize the findings that have helped to provide our most definitive understanding of the selective regulation of cAMP signalling by SOCE.
Asunto(s)
Señalización del Calcio , AMP Cíclico/metabolismo , Complejos Multiproteicos/metabolismo , Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Humanos , Unión ProteicaRESUMEN
Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca(2+). However, whether AKAPs play a role in the control of AC activity by Ca(2+) is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca(2+)-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca(2+) events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca(2+)-stimulated cAMP production.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/metabolismo , Calcio/metabolismo , AMP Cíclico/biosíntesis , Células Secretoras de Insulina/metabolismo , Neuronas/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Adenilil Ciclasas/genética , Animales , Animales Recién Nacidos , Western Blotting , Calcio/farmacología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuronas/citología , Unión Proteica , Interferencia de ARN , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Spatial and temporal compartmentalization of cAMP (and its target proteins) is central to the ability of this second messenger to govern cellular activity over timescales ranging from milliseconds to several hours. Recent years have witnessed a burgeoning of methodologies that enable researchers to directly monitor rapid subcellular cAMP dynamics, which are unobtainable by traditional cAMP assays. In this review, we examine cAMP biosensors that are currently available for measuring cAMP at the single-cell level, compare their various operating principles and discuss their applications.
Asunto(s)
Bioensayo/métodos , Técnicas Biosensibles/métodos , Calcio/metabolismo , Fenómenos Fisiológicos Celulares , AMP Cíclico/metabolismo , Perfilación de la Expresión Génica/métodos , Microscopía Fluorescente/métodosRESUMEN
Capacitative Ca(2+) entry (CCE), which occurs through the plasma membrane as a result of Ca(2+) store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca(2+) store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca(2+) entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca(2+)-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca(2+) entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca(2+) entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca(2+)-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.
Asunto(s)
Adenilil Ciclasas/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Adenilil Ciclasas/fisiología , Animales , Canales de Calcio/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Ratas , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/fisiologíaRESUMEN
Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Citosol/metabolismo , Espacio Intracelular/enzimología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Extractos Celulares , Línea Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/química , Citosol/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Espacio Intracelular/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ratones , Transporte de Proteínas/efectos de los fármacos , Ratas , Fracciones Subcelulares/efectos de los fármacos , Xantinas/farmacologíaRESUMEN
The interplay between calcium ion (Ca(2+)) and cyclic adenosine monophosphate (cAMP) signaling underlies crucial aspects of cell homeostasis. The membrane-bound Ca(2+)-regulated adenylyl cyclases (ACs) are pivotal points of this integration. These enzymes display high selectivity for Ca(2+) entry arising from the activation of store-operated Ca(2+) (SOC) channels, and they have been proposed to functionally colocalize with SOC channels to reinforce crosstalk between the two signaling pathways. Using a multidisciplinary approach, we have identified a direct interaction between the amino termini of Ca(2+)-stimulated AC8 and Orai1, the pore component of SOC channels. High-resolution biosensors targeted to the AC8 and Orai1 microdomains revealed that this protein-protein interaction is responsible for coordinating subcellular changes in both Ca(2+) and cAMP. The demonstration that Orai1 functions as an integral component of a highly organized signaling complex to coordinate Ca(2+) and cAMP signals underscores how SOC channels can be recruited to maximize the efficiency of the interplay between these two ubiquitous signaling pathways.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/metabolismo , Canales de Calcio/química , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Homeostasis , Humanos , Iones , Proteínas Luminiscentes/metabolismo , Proteína ORAI1 , Estructura Terciaria de ProteínaRESUMEN
The adenylyl cyclases are variously regulated by G protein subunits, a number of serine/threonine and tyrosine protein kinases, and Ca(2+). In some physiological situations, this regulation can be readily incorporated into a hormonal cascade, controlling processes such as cardiac contractility or neurotransmitter release. However, the significance of some modes of regulation is obscure and is likely only to be apparent in explicit cellular contexts (or stages of the cell cycle). The regulation of many of the ACs by the ubiquitous second messenger Ca(2+) provides an overarching mechanism for integrating the activities of these two major signaling systems. Elaborate devices have been evolved to ensure that this interaction occurs, to guarantee the fidelity of the interaction, and to insulate the microenvironment in which it occurs. Subcellular targeting, as well as a variety of scaffolding devices, is used to promote interaction of the ACs with specific signaling proteins and regulatory factors to generate privileged domains for cAMP signaling. A direct consequence of this organization is that cAMP will exhibit distinct kinetics in discrete cellular domains. A variety of means are now available to study cAMP in these domains and to dissect their components in real time in live cells. These topics are explored within the present review.
Asunto(s)
Adenilil Ciclasas/metabolismo , Señalización del Calcio/fisiología , AMP Cíclico/metabolismo , Animales , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.
Asunto(s)
AMP Cíclico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Prostaglandina/metabolismo , Sistemas de Mensajero Secundario/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , Línea Celular , AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo/fisiología , Genes Dominantes , Humanos , Interferencia de ARN , Receptores Adrenérgicos beta 2/genética , Receptores de Prostaglandina/genética , beta-ArrestinasRESUMEN
The spatial and temporal complexity of Ca2+ signalling is central to the regulation of a diverse range of cellular processes. The decoding of dynamic Ca2+ signals is, in part, mediated by the ability of Ca2+ to regulate other second messengers, including cyclic AMP (cAMP). A number of kinetic models (including our own) predict that interdependent Ca2+ and cAMP oscillations can be generated. A previous study in Xenopus neurons illustrated prolonged, low-frequency cAMP oscillations during bursts of Ca2+ transients. However, the detection of more dynamic Ca2+ driven changes in cAMP has, until recently, been limited by the availability of suitable cAMP probes with high temporal resolution. We have used a newly developed FRET-based cAMP indicator comprised of the cAMP binding domain of Epac-1 to examine interplay between Ca2+ and cAMP dynamics. This probe was recently used in excitable cells to reveal an inverse relationship between cAMP and Ca2+ oscillations as a consequence of Ca2+-dependent activation of phosphodiesterase 1 (PDE1). Here, we have used human embryonic kidney (HEK293) cells expressing the type 8 adenylyl cyclase (AC8) to examine whether dynamic Ca2+ changes can mediate phasic cAMP oscillations as a consequence of Ca2+-stimulated AC activity. During artificial or agonist-induced Ca2+ oscillations we detected fast, periodic changes in cAMP that depended upon Ca2+ stimulation of AC8 with subsequent PKA-mediated phosphodiesterase 4 (PDE4) activity. Carbachol (10 microM) evoked cAMP transients with a peak frequency of approximately 3 minute(-1), demonstrating phasic oscillations in cAMP and Ca2+ in response to physiological stimuli. Furthermore, by imposing a range of Ca2+-oscillation frequencies, we demonstrate that AC8 acts as a low-pass filter for high-frequency Ca2+ events, enhancing the regulatory options available to this signalling pathway.
Asunto(s)
Adenilil Ciclasas/metabolismo , Relojes Biológicos/fisiología , Calcio/farmacología , AMP Cíclico/metabolismo , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/genética , Alprostadil/farmacología , Relojes Biológicos/efectos de los fármacos , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Carbacol/farmacología , Catálisis , Línea Celular , Células Cultivadas , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Humanos , Isoproterenol/farmacología , Factores de TiempoRESUMEN
The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Sistemas de Mensajero Secundario/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Proteínas de Anclaje a la Quinasa A , Alprostadil/metabolismo , Animales , Técnicas Biosensibles , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Complejos Multiproteicos , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Ratas , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismoRESUMEN
Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Here, we provide evidence for interactions between AKAP79/150 and ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/biosíntesis , Isoenzimas/metabolismo , Proteínas de Anclaje a la Quinasa A , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Activación Enzimática , Retroalimentación Fisiológica , Humanos , Modelos Biológicos , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca2+ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH(i)), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH(i) on Ca2+-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na+/H+ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH(i) is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Concentración de Iones de Hidrógeno , Isoenzimas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adenilil Ciclasas/genética , Animales , Caveolinas/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Humanos , Isoenzimas/genética , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Neuronal electrical activity causes only modest changes in global intracellular pH (pH(i)). We have measured regional pH(i) differences in isolated patch-clamped neurones during depolarization, using confocal imaging of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescence. The pH(i) shifts in the soma were as expected; however, substantially larger shifts occurred in other regions. These regional differences were still observed in the presence of CO(2)-HCO(3)(-), they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 +/- 1.3 % (n = 9; approximately 0.1 pH unit acidification) following a 1 s depolarization to 0 mV; this was more than 4-fold greater than the relative shift seen in the soma. Depolarization to +40 mV for 1 s caused a 46.7 +/- 7.0 % increase (n = 11; approximately 0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6-fold that seen in the soma. Application of 5 % CO(2)-20 mM HCO(3)(-) did not significantly reduce the size of the +40 mV-evoked local pH shifts despite carbonic anhydrase activity. The pH(i) gradient between regions approximately 50 microm apart, resulting from acid shifts, took 10.3 +/- 3.1 s (n = 6) to decay by 50 %, whereas the pH(i) gradient resulting from alkaline shifts took only 3.7 +/- 1.4 s (n = 12) to decay by 50 %. The regional rates of acidification and calcium recovery were closely related, suggesting that the acidic pH microdomains resulted from Ca(2+)-H(+) pump activity. The alkaline pH microdomains were blocked by zinc and resulted from proton channel opening. It is likely that the microdomains result from transmembrane acid fluxes in areas with different surface area to volume ratios. Such neuronal pH microdomains are likely to have consequences for local receptor, channel and enzyme function in restricted regions.
Asunto(s)
Calcio/metabolismo , Hidrógeno/metabolismo , Neuronas/fisiología , Animales , Arilsulfonatos , Bicarbonatos/farmacología , Calibración , Dióxido de Carbono/farmacología , Anhidrasas Carbónicas/metabolismo , Separación Celular , Electrofisiología , Colorantes Fluorescentes , Caracoles Helix , Concentración de Iones de Hidrógeno/efectos de los fármacosRESUMEN
Our aim was to test the hypothesis that depolarization-induced intracellular pH (pH(i)) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH-sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole-cell patch-clamped rat cerebellar Purkinje cells. In the absence of added CO(2)-HCO(3)(-), depolarization to +20 mV for 1 s caused large (approximately 0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (approximately 0.06 pH units) and slower. The pH(i) shifts were smaller in the presence of 5 % CO(2)-25 mM HCO(3)(-)-buffered saline (approximately 0.08 pH units in the dendrites and approximately 0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 microM) doubled the size of the dendritic acid shifts in the presence of CO(2)-HCO(3)(-), indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mM) inhibited the depolarization-evoked acid shifts. We investigated more physiological pH(i) changes by evoking modest bursts of action potentials (approximately 10 s duration) in CO(2)-HCO(3)(-)-buffered saline. Such neuronal firing induced an acidification of approximately 0.11 pH units in the fine dendritic regions, but only approximately 0.03 pH units in the soma. There was considerable variation in the size of the pH(i) shifts between cells, with dendritic acid shifts as large as 0.2-0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pH(i) changes (pH microdomains) might act as important signals in synaptic function.