RESUMO
The primary cilium permits compartmentalization of specific signaling pathways, including elements of the Hedgehog (Hh) pathway. Hh transcriptional activity is thought to be negatively regulated by constitutively high ciliary cAMP maintained by the Gα(s)-coupled GPCR, GPR161. However, cilia also sequester many other Gα(s)-coupled GPCRs with unknown potential to regulate Hh. Here we used biosensors optimized for ciliary cAMP and strategies to isolate signals in the cilium from the cell body and neighboring cells. We found that ciliary cAMP was not elevated relative to cellular cAMP, inconsistent with constitutive cAMP production. Gα(s)-coupled GPCRs (e.g., the 5-HT6 serotonin and D1R dopamine receptor) had reduced ability to generate cAMP upon trafficking to the ciliary membrane. However, activation of the Hh pathway restored or amplified GPCR function to permit cAMP elevation selectively in the cilium. Hh therefore enables its own local GPCR-dependent cAMP regulatory circuit. Considering that GPCRs comprise much of the druggable genome, these data suggest alternative strategies to modify Hh signaling.
Assuntos
Cílios/metabolismo , AMP Cíclico/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Animais , Linhagem Celular , Camundongos , Células NIH 3T3 , Receptores Dopaminérgicos/metabolismo , Serotonina/metabolismoRESUMO
Apical cAMP-dependent CFTR Cl(-) channels are essential for efficient vectorial movement of ions and fluid into the lumen of the colon. It is well known that Ca(2+)-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca(2+), and the existence of apical Ca(2+)-dependent Cl(-) channels in the native colonic epithelium is controversial, leaving the identity of the Ca(2+)-activated component unresolved. We recently showed that decreasing free Ca(2+) concentration ([Ca(2+)]) within the endoplasmic reticulum (ER) lumen elicits a rise in intracellular cAMP. This process, which we termed "store-operated cAMP signaling" (SOcAMPS), requires the luminal ER Ca(2+) sensor STIM1 and does not depend on changes in cytosolic Ca(2+). Here we assessed the degree to which SOcAMPS participates in Ca(2+)-activated Cl(-) transport as measured by transepithelial short-circuit current (Isc) in polarized T84 monolayers in parallel with imaging of cAMP and PKA activity using fluorescence resonance energy transfer (FRET)-based reporters in single cells. In Ca(2+)-free conditions, the Ca(2+)-releasing agonist carbachol and Ca(2+) ionophore increased Isc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca(2+) chelator BAPTA-AM. The effect on Isc was enhanced in the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), inhibited by the CFTR inhibitor CFTRinh-172 and the PKA inhibitor H-89, and unaffected by Ba(2+) or flufenamic acid. We propose that a discrete component of the "Ca(2+)-dependent" secretory activity in the colon derives from cAMP generated through SOcAMPS. This alternative mode of cAMP production could contribute to the actions of diverse xenobiotic agents that disrupt ER Ca(2+) homeostasis, leading to diarrhea.
Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Colo/metabolismo , AMP Cíclico/metabolismo , Linhagem Celular Tumoral , Colo/citologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Transdução de SinaisRESUMO
Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca(2+) ] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this 'store-operated' pathway requires the ER Ca(2+) sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca(2+) entry via the plasma membrane Ca(2+) channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca(2+) /cAMP crosstalk system.
Assuntos
Adenilil Ciclases/metabolismo , Canais de Cálcio/metabolismo , AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Adenilil Ciclases/genética , Técnicas Biossensoriais , Cálcio/metabolismo , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Toxina Pertussis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Molécula 1 de Interação Estromal , Molécula 2 de Interação EstromalRESUMO
Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid abundant in fish oil that exerts a wide spectrum of documented beneficial health effects in humans. Because dietary interventions are relatively inexpensive and are widely assumed to be safe, they have broad public appeal. Their endorsement can potentially have a major impact on human health, but hard mechanistic evidence that specifies how these derivatives work at the cellular level is limited. EPA (50 microM) caused a small elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)]) in intact NCM460 human colonic epithelial cells as measured by fura 2 and a profound drop of [Ca(2+)] within the endoplasmic reticulum (ER) of permeabilized cells as monitored by compartmentalized mag-fura 2. Total internal reflection fluorescence microscopy showed that this loss of ER store [Ca(2+)] led to translocation of the ER-resident transmembrane Ca(2+) sensor STIM1. Using sensitive FRET-based sensors for cAMP in single cells, we further found that EPA caused a substantial increase in cellular cAMP concentration, a large fraction of which was dependent on the drop in ER [Ca(2+)], but independent of cytosolic Ca(2+). An additional component of the EPA-induced cAMP signal was sensitive to the phosphodiesterase inhibitor isobutyl methylxanthine. We conclude that EPA slowly releases ER Ca(2+) stores, resulting in the generation of cAMP. The elevated cAMP is apparently independent of classical G protein-coupled receptor activation and is likely the consequence of a newly described "store-operated" cAMP signaling pathway that is mediated by STIM1.
Assuntos
Colo/citologia , AMP Cíclico/metabolismo , Ácido Eicosapentaenoico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , 1-Metil-3-Isobutilxantina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Colo/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Molécula 1 de Interação EstromalRESUMO
Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (I(sc)), transepithelial potential (V(t)) and resistance (R(t)) were recorded in the continuous presence of cadmium. Addition of cadmium (20 microM to 1mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in I(sc) cannot be explained by an action on: 1) H(2) histamine receptor, 2) Ca(2+) signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H(+)/K(+)-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H(+)/K(+)-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.
Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Ácido Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Anidrases Carbônicas/metabolismo , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Etilenodiaminas/farmacologia , Mucosa Gástrica/metabolismo , Histamina/farmacologia , Técnicas In Vitro , Omeprazol/farmacologia , Inibidores da Bomba de Prótons , Rana esculentaRESUMO
Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/farmacologia , Receptores de Detecção de Cálcio/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Técnicas Biossensoriais/métodos , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eritromicina/análogos & derivados , Eritromicina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Toxina Pertussis/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
It is generally assumed that the functional consequences of stimulation with Ca2+ -mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ "sensor" raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a "third messenger" via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+.
Assuntos
Adenina/análogos & derivados , Cálcio/metabolismo , Carbacol/farmacologia , Mucosa Gástrica/patologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adenina/farmacologia , Animais , Eletrodos , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Mucosa Gástrica/metabolismo , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Pepsinogênio A/química , Pepsinogênio A/metabolismo , Ranidae , Transdução de Sinais , Espermina/metabolismoRESUMO
Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.
Assuntos
AMP Cíclico/metabolismo , Imagem Óptica/métodos , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Genes Reporter , Humanos , Microdomínios da Membrana/metabolismoRESUMO
The extracellular calcium-sensing receptor (CaR), a ubiquitous class C G-protein-coupled receptor (GPCR), is responsible for the control of calcium homeostasis in body fluids. It integrates information about external Ca 2+ and a surfeit of other endogenous ligands into multiple intracellular signals, but how is this achieved? This review will focus on some of the exciting concepts in CaR signaling and pharmacology that have emerged in the last few years.
RESUMO
In multicellular organisms, cells are crowded together in organized communities, surrounded by an interstitial fluid of extremely limited volume. Local communication between adjacent cells is known to occur through gap junctions in cells that are physically connected, or through the release of paracrine signaling molecules (e.g. ATP, glutamate, nitric oxide) that diffuse to their target receptors through the extracellular microenvironment. Recent evidence hints that calcium ions may possibly be added to the list of paracrine messengers that allow cells to communicate with one another. Local fluctuations in extracellular [Ca2+] can be generated as a consequence of intracellular Ca2+ signaling events, owing to the activation of Ca2+ influx and efflux pathways at the plasma membrane. In intact tissues, where the interstitial volumes between cells are much smaller than the cells themselves, this can result in significant alterations in external [Ca2+]. This article will explore emerging evidence that these extracellular [Ca2+] changes can be detected by the extracellular calcium-sensing receptor (CaR) on adjacent cells, forming the basis for a paracrine signaling system. Such a mechanism could potentially provide CaR-expressing cells with the means to sense the Ca2+ signaling status of their neighbors, and expand the utility of the intracellular Ca2+ signal to a domain outside the cell.
Assuntos
Comunicação Celular/fisiologia , Epitélio/fisiologia , Receptores de Detecção de Cálcio/fisiologia , Animais , Comunicação Autócrina/fisiologia , Cálcio/análise , Cálcio/fisiologia , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Modelos BiológicosRESUMO
BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP). METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge") was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.
Assuntos
Soluções Tampão , Proteínas Quinases Dependentes de AMP Cíclico/química , AMP Cíclico/química , Técnicas Biossensoriais , Linhagem Celular , Clonagem Molecular , Transferência Ressonante de Energia de Fluorescência , Junções Comunicantes , Regulação da Expressão Gênica , Células HeLa , Humanos , Mutação , Mutação Puntual , Sistemas do Segundo Mensageiro/genética , Transdução de SinaisRESUMO
Depletion of Ca(2+) from the endoplasmic reticulum (ER) results in activation of plasma membrane Ca(2+) entry channels. This 'store-operated' process requires translocation of a transmembrane ER Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to sites closely apposed to Ca(2+) channels at the cell surface. However, it is not known whether a reduction in Ca(2+) stores is coupled to other signalling pathways by this mechanism. We found that lowering the concentration of free Ca(2+) in the ER, independently of the cytosolic Ca(2+) concentration, also led to recruitment of adenylyl cyclases. This resulted in enhanced cAMP accumulation and PKA activation, measured using FRET-based cAMP indicators. Translocation of STIM1 was required for efficient coupling of ER Ca(2+) depletion to adenylyl cyclase activity. We propose the existence of a pathway (store-operated cAMP signalling or SOcAMPS) in which the content of internal Ca(2+) stores is directly connected to cAMP signalling through a process that involves STIM1.
Assuntos
Sinalização do Cálcio , AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Adenilil Ciclases/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quelantes/farmacologia , AMP Cíclico/biossíntese , Citosol/efeitos dos fármacos , Citosol/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transporte Proteico/efeitos dos fármacos , Molécula 1 de Interação EstromalRESUMO
The mechanisms for the formation of the osmotic gradient driving water movements in the gastric gland and its modulation via the extracellular Ca(2+)-sensing receptor (CaR) were investigated. Real time measurements of net water flux in the lumen of single gastric glands of the intact amphibian stomach were performed using ion-selective double-barreled microelectrodes. Water movement was measured by recording changes in the concentration of impermeant TEA(+) ions ([TEA(+)](gl)) with TEA(+)-sensitive microelectrodes inserted in the lumen of individual gastric glands. Glandular K(+) (K(+)(gl)) and H(+) (pH(gl)) were also measured by using K(+)- and H(+)-sensitive microelectrodes, respectively. Stimulation with histamine significantly decreased [TEA](gl), indicating net water flow toward the gland lumen. This response was inhibited by the H(+)/K(+)-ATPase inhibitor, SCH 28080. Histamine also elicited a significant and reversible increase in [K(+)](gl) that was blocked by chromanol 293B, a blocker of KCQN1 K(+) channels. Histamine failed to induce net water flow in the presence of chromanol 293B. In the "resting state," stimulation of CaR with diverse agonists resulted in significant increase in [TEA](gl). CaR activation also significantly reduced histamine-induced water secretion and apical K(+) transport. Our data validate the strong link between histamine-stimulated acid secretion and water transport. We also show that cAMP-dependent [K(+)](gl) elevation prior to the onset of acid secretion generates the osmotic gradient initially driving water into the gastric glands and that CaR activation inhibits this process, probably through reduction of intracellular cAMP levels.
Assuntos
Proteínas de Anfíbios/metabolismo , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Água/metabolismo , Proteínas de Anfíbios/antagonistas & inibidores , Animais , Cromanos/farmacologia , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Histamina/farmacologia , Imidazóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Microeletrodos , Osmose/efeitos dos fármacos , Osmose/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores da Bomba de Prótons , Rana esculenta , Receptores de Detecção de Cálcio/antagonistas & inibidores , Sulfonamidas/farmacologiaRESUMO
BACKGROUND & AIMS: Secondary bile acids like deoxycholic acid (DCA) are well-established tumor promoters that may exert their pathologic actions by interfering with intracellular signaling cascades. METHODS: We evaluated the effects of DCA on Ca2+ signaling in BHK-21 fibroblasts using fura-2 and mag-fura-2 to measure cytoplasmic and intraluminal internal stores [Ca2+], respectively. Furthermore, green fluorescent protein (GFP)-based probes were used to monitor time courses of phospholipase C (PLC) activation (pleckstrin-homology [PH]-PLCdelta-GFP), and translocation of protein kinase C (PKC) and a major PKC substrate, myristolated alanine-rich C-kinase substrate (MARCKS). RESULTS: DCA (50-250 micromol/L) caused profound Ca2+ release from intracellular stores of intact or permeabilized cells. Correspondingly, DCA increased cytoplasmic Ca2+ to levels that were approximately 120% of those stimulated by Ca2+-mobilizing agonists in the presence of external Ca2+, and approximately 60% of control in Ca2+-free solutions. DCA also caused dramatic translocation of PH-PLCdelta-GFP, and conventional, Ca2+/diacylglycerol (DAG)-dependent isoforms of PKC (PKC-betaI and PKC-alpha), and MARCKS-GFP, but only in Ca2+-containing solutions. DCA had no effect on localization of a novel (PKCdelta) or an atypical (PKCzeta) PKC isoform. CONCLUSIONS: Data are consistent with a model in which DCA directly induces both Ca2+ release from internal stores and persistent Ca2+ entry at the plasma membrane. The resulting microdomains of high Ca2+ levels beneath the plasma membrane appear to directly activate PLC, resulting in modest InsP 3 and DAG production. Furthermore, the increased Ca2+ entry stimulates vigorous recruitment of conventional PKC isoforms to the plasma membrane.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Ácido Desoxicólico/farmacologia , Fibroblastos/metabolismo , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Citoplasma/metabolismo , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Humanos , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Substrato Quinase C Rico em Alanina MiristoiladaRESUMO
Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 microM TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps.