RESUMEN

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss. The neovascular or "wet" form of AMD can be treated to varying degrees with anti-angiogenic drugs, but geographic atrophy (GA) is an advanced stage of the more prevalent "dry" form of AMD for which there is no effective treatment. Development of GA has been linked to loss of the microRNA (miRNA)-processing enzyme DICER1 in the mature retinal pigmented epithelium (RPE). This loss results in the accumulation of toxic transcripts of Alu transposable elements, which activate the NLRP3 inflammasome and additional downstream pathways that compromise the integrity and function of the RPE. However, it remains unclear whether the loss of miRNA processing and subsequent gene regulation in the RPE due to DICER1 deficiency also contributes to RPE cell death. To clarify the role of miRNAs in RPE cells, we used two different mature RPE cell-specific Cre recombinase drivers to inactivate either Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), thus removing RPE miRNA regulatory activity in mice by disrupting two independent and essential steps of miRNA biogenesis. In contrast with prior studies, we found that the loss of each factor independently led to strikingly similar defects in the survival and function of the RPE and retina. These results suggest that the loss of miRNAs also contributes to RPE cell death and loss of visual function and could affect the pathology of dry AMD.

Asunto(s)

ARN Helicasas DEAD-box/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Epitelio Pigmentado de la Retina/citología , Ribonucleasa III/metabolismo , Animales , Supervivencia Celular , ARN Helicasas DEAD-box/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagosomas/metabolismo , Fagosomas/patología , Proteínas de Unión al ARN/genética , Retina , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Ribonucleasa III/genética

RESUMEN

MicroRNAs (miRNAs) regulate multiple genes, often within the same pathway, fine-tuning expression of key factors and stabilizing gene networks against aberrant fluctuations. The demanding physiologic functions of photoreceptor cells and the retinal pigmented epithelium necessitate precise gene regulation to maintain their homeostasis and function, thus rendering these postmitotic cells vulnerable to premature death in retinal degenerative disorders. Recent studies of the physiologic impact of miRNAs in these cells clearly demonstrate that miRNAs are an essential component of that gene regulation. These important advances provide the foundation for future exploration of miRNA-regulated gene networks in the eye to facilitate the development of miRNA-targeted therapeutics to combat blinding diseases.

Asunto(s)

Diferenciación Celular/genética , Regulación de la Expresión Génica/fisiología , MicroARNs/genética , Retina/metabolismo , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Humanos

RESUMEN

BACKGROUND: Though accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1 (GFP/GFP) and Ccr2 (RFP/RFP) mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation. METHODS: In this study, c-mer proto-oncogene tyrosine kinase (Mertk) (-/-) mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were established by breeding Mertk (-/-) , Cx3cr1 (GFP/GFP) , and Ccr2 (RFP/RFP) mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry. RESULTS: Four-week-old Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. These invading cells were still observed in the subretinal space in 18-week-old animals. CONCLUSIONS: Cx3cr1-GFP-positive microglia and Ccr2-RFP-positive macrophages were distinguishable in the retinas of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. In addition, Ccr2 expression in Cx3cr1 positive microglia is a feature of microglial activation in RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice enabled observation of microglial activation over time during RD and may be useful for developing inflammation-targeted treatment strategies for RD in the future.

Asunto(s)

Regulación de la Expresión Génica/genética , Receptores CCR2/metabolismo , Receptores de Quimiocina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Animales , Receptor 1 de Quimiocinas CX3C , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Leucocitos/metabolismo , Leucocitos/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Mutación/genética , Neuronas/metabolismo , Neuronas/patología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptores CCR2/genética , Receptores de Quimiocina/genética , Retina/metabolismo , Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Factores de Tiempo , Tirosina Quinasa c-Mer

RESUMEN

Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases.

Asunto(s)

Proteínas Argonautas/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoprecipitación/métodos , MicroARNs/genética , Retina/fisiología , Animales , Bovinos , Regulación de la Expresión Génica

RESUMEN

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.

Asunto(s)

ARN Helicasas DEAD-box/fisiología , MicroARNs/fisiología , Degeneración Retiniana/etiología , Células Fotorreceptoras Retinianas Bastones/patología , Ribonucleasa III/fisiología , Edad de Inicio , Animales , Ciclo Celular , Supervivencia Celular , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Electrorretinografía , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Técnicas de Placa-Clamp , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Análisis de Secuencia de ARN , Tomografía de Coherencia Óptica , Visión Ocular

RESUMEN

PURPOSE: Determine the impact of rod photoreceptor-specific expression of Cre recombinase on the kinetics of phototransduction in the mouse eye and identify changes in gene expression that underlie any observed phenotypic differences. METHODS: Transretinal ERG and single-cell suction electrode recordings were used to measure the kinetics of phototransduction in a mouse line exhibiting rod photoreceptor-specific Cre recombinase expression, and the results were compared with those from control non-Cre-expressing littermates. Gene expression changes were evaluated using RNA sequencing transcriptome analysis. The pattern of expression of Rgs9bp was determined by mapping sequencing reads to the mouse genome and performing 3'-rapid amplification of cDNA ends (3'-RACE). RESULTS: Expression of the rod-specific iCre75 transgene was accompanied by accelerated phototransduction inactivation, likely due to overexpression of the Rgs9bp gene, which encodes the Rgs9 anchor protein (R9AP). R9AP upregulation stabilized the RGS9 GAP complex, altering phototransduction kinetics. 3'-Race identified an abundant, unexpected Rgs9bp-Prm1 fusion mRNA in Cre-expressing mouse retinas, which was determined to be derived from a second transgene present in the iCre75 line. CONCLUSIONS: Here we report the presence of a second, R9AP-expressing transgene in the iCre75 mouse line, leading to altered kinetics of phototransduction. These results highlight an important caveat that must be considered when utilizing this mouse line for rod photoreceptor-specific gene loss of function studies.

Asunto(s)

Regulación de la Expresión Génica , Fototransducción/genética , Proteínas de la Membrana/genética , ARN Mensajero/genética , Retina/metabolismo , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo , Immunoblotting , Integrasas/genética , Integrasas/metabolismo , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Retina/citología , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo

RESUMEN

microRNAs (miRNAs) are small, stable RNA molecules that post-transcriptionally regulate gene expression in plants and animals by base pairing to partially complementary sequences on target mRNAs to inhibit protein synthesis. More than 250 miRNAs are reportedly expressed in the retina, and miRNA gene regulation has been shown to affect retinal development, function, and disease. Here we highlight recent advances in understanding the functional roles of vertebrate retinal miRNAs. Details are emerging about the physiological impact of specific miRNAs in the developing and mature retina, and we discuss a group of emerging technologies for studying miRNAs, which can be employed to yield a deeper understanding of retinal miRNA gene regulation.

Asunto(s)

Regulación de la Expresión Génica , MicroARNs/genética , Retina/crecimiento & desarrollo , Retina/metabolismo , Animales , Humanos , MicroARNs/metabolismo

RESUMEN

The discovery of microRNA (miRNA)-mediated gene silencing has added a new level of complexity to our understanding of post-transcriptional control of gene expression. Considering the ubiquity of miRNA-mediated repression throughout basic cellular processes, understanding its mechanism of action is paramount to obtain a clear picture of the regulation of gene expression in biological systems. Although many miRNAs and their targets have been identified, a detailed understanding of miRNA action remains elusive. miRNAs regulate gene expression at the post-transcriptional level, through both translational inhibition and mRNA destabilization. Recent reports suggest that many miRNA effects are mediated through proteins of the GW182 family. This chapter focuses on the multiple and potentially overlapping mechanisms that miRNAs utilize to regulate gene expression in eukaryotes.

Asunto(s)

Regulación de la Expresión Génica , MicroARNs/metabolismo , Transcripción Genética , Animales , Células Eucariotas/metabolismo , Humanos , MicroARNs/biosíntesis , Biosíntesis de Proteínas/genética

RESUMEN

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

Asunto(s)

Silenciador del Gen , MicroARNs/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Animales , Proteínas Argonautas , Ascitis/genética , Ascitis/metabolismo , Autoantígenos/metabolismo , Sitios de Unión , Carcinoma Krebs 2/genética , Carcinoma Krebs 2/metabolismo , Sistema Libre de Células , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Exorribonucleasas , Células HeLa , Humanos , Cinética , Ratones , Proteínas de Unión a Poli(A)/genética , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas/genética , Estabilidad del ARN , Complejo Silenciador Inducido por ARN/genética , Receptores CCR4/metabolismo , Proteínas Represoras , Ribonucleasas , Transfección

RESUMEN

Studies in eukaryotes and prokaryotes have revealed that gene expression is not only controlled through altering the rate of transcription but also through varying rates of translation and mRNA decay. Indeed, the expression level of a protein is strongly affected by the steady state level of its mRNA. RNA decay can, along with transcription, play an important role in regulating gene expression by fine-tuning the steady state level of a given transcript and affecting its subsequent decoding during translation. Alterations in mRNA stability can in turn have dramatic effects on cell physiology and as a consequence the fitness and survival of the organism. Recent evidence suggests that mRNA decay can be regulated in response to environmental cues in order to enable the organism to adapt to its changing surroundings. Bacteria have evolved unique post transcriptional control mechanisms to enact such adaptive responses through: 1) general mRNA decay, 2) differential mRNA degradation using small non-coding RNAs (sRNAs), and 3) selective mRNA degradation using the tmRNA quality control system. Here, we review our current understanding of these molecular mechanisms, gleaned primarily from studies of the model gram negative organism Escherichia coli, that regulate the stability and degradation of normal and defective transcripts.

Asunto(s)

Bacterias/genética , Procesamiento Postranscripcional del ARN/fisiología , ARN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Control de Calidad , Estabilidad del ARN , ARN Bacteriano/genética , Transcripción Genética

RESUMEN

In bacteria, ribosomes stalled at the 3'-end of nonstop or defective mRNAs are rescued by the action of a specialized ribonucleoprotein complex composed of tmRNA and SmpB protein in a process known as trans-translation; for recent reviews see Dulebohn et al. [2007], Keiler [2007], and Moore and Sauer [2007]. tmRNA is a bifunctional RNA that acts as both a tRNA and an mRNA. SmpB-bound tmRNA is charged with alanine by alanyl-tRNA synthetase and recognized by EF-Tu (GTP). The quaternary complex of tmRNA-SmpB-EF-Tu and GTP recognizes stalled ribosomes and transfers the nascent polypeptide to the tRNA-like domain of tmRNA. A specialized reading frame within tmRNA is then engaged as a surrogate mRNA to append a 10 amino acid (ANDENYALAA) tag to the C-terminus of the nascent polypeptide. A stop codon at the end of the tmRNA reading frame then facilitates normal termination and recycling of the translation machinery. Through this surveillance mechanism, stalled ribosomes are rescued, and nascent polypeptides bearing the C-terminal tmRNA-tag are directed for proteolysis. Several proteases (ClpXP, ClpAP, Lon, FtsH, and Tsp) are known to be involved in the degradation of tmRNA-tagged proteins (Choy et al., 2007; Farrell et al., 2005; Gottesman et al., 1998; Herman et al., 1998, 2003; Keiler et al., 1996). In addition to its ribosome rescue and peptide tagging activities, trans-translation also facilitates the selective decay of nonstop mRNAs in a process that is dependent on the activities of SmpB protein, tmRNA, and the 3' to 5'-exonuclease, RNase R (Mehta et al., 2006; Richards et al., 2006; Yamamoto et al., 2003). Here, we describe methods and strategies for the purification of tmRNA, SmpB, Lon, and RNase R from Escherichia coli that are likely to be applicable to other bacterial species. Protocols for the purification of the Clp proteases, Tsp, and FtsH, as well as EF-Tu and other essential E. coli translation factors may be found elsewhere (Joshi et al., 2003; Kihara et al., 1996; Makino et al., 1999; Maurizi et al., 1990; Shotland et al., 2000). In addition, we present biochemical and genetic assays to study the various aspects of the trans-translation mechanism.

Asunto(s)

ARN Bacteriano/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Bacteriano/química , ARN Bacteriano/aislamiento & purificación , ARN Bacteriano/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación

RESUMEN

Small protein B (SmpB) is a requisite component of the transfer messenger RNA (tmRNA)-mediated bacterial translational quality control system known as trans-translation. The initial binding of tmRNA and its subsequent accommodation into the ribosomal A-site are activities intimately linked to SmpB protein function. From a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does SmpB pre-bind ribosomes to recruit tmRNA. We have assessed, both in vivo and in vitro, the nature and stability of free SmpB interactions with stalled ribosomes and examined whether these interactions are functionally relevant. We present evidence to demonstrate that interaction of free SmpB with ribosomes is salt sensitive and significantly more labile than interaction of the SmpB.tmRNA complex with ribosomes. Upon dissociation of 70 S ribosomes SmpB partitions primarily with tmRNA rather than ribosomal subunits. This finding is consistent with biochemical and structural data demonstrating that tmRNA is the high-affinity binding partner of SmpB. Moreover, we show that under normal physiological conditions roughly similar numbers of SmpB and tmRNA molecules are present in cells. Our investigations also reveal that upon induction of a nonstop mRNA, SmpB is enriched in stalled ribosome fractions only in the presence of tmRNA. Based on these findings, we conclude that SmpB does not pre-bind stalled ribosome and that functional SmpB-stalled ribosome interactions require tmRNA. We propose that a 1:1:1 complex of SmpB.tmRNA.EF-Tu(GTP) recognizes and binds a stalled ribosome to initiate trans-translation.

Asunto(s)

ARN Bacteriano/química , Proteínas de Unión al ARN/química , Bioquímica/métodos , Microscopía por Crioelectrón , Relación Dosis-Respuesta a Droga , Guanosina Trifosfato/química , Biosíntesis de Proteínas , ARN/metabolismo , Proteínas Ribosómicas/química , Subunidades Ribosómicas , Ribosomas/química , Sales (Química)/farmacología , Thermus/metabolismo

RESUMEN

The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality-control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and a mRNA. The tmRNA system orchestrates three key biological functions: (1) recognition and rescue of ribosomes stalled on aberrant mRNAs, (2) disposal of the causative defective mRNAs, and (3) addition of a degradation tag to ribosome-associated protein fragments for directed proteolysis. Although not essential in Escherichia coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence suggests that in addition to its quality-control function the tmRNA system might also play a key regulatory role in certain physiological pathways. This review will focus on recent advances in our understanding of the structural properties, mechanistic details, and physiological significance of this unique RNA and its principal protein partners.

Asunto(s)

Proteínas Bacterianas/metabolismo , ARN Bacteriano/fisiología , ARN Mensajero/fisiología , ARN de Transferencia/fisiología , Ribosomas/fisiología , Secuencia de Bases , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Biosíntesis de Proteínas/fisiología , ARN Bacteriano/química , Proteínas de Unión al ARN/metabolismo

RESUMEN

SsrA is a versatile RNA molecule found in all bacteria that functions as both a tRNA and an mRNA. SsrA rescues ribosomes stalled on damaged mRNAs and directs the tagging and degradation of their aberrant protein products. Small protein B (SmpB) is required for all known activities of SsrA. The two known functions of SmpB are binding SsrA RNA and promoting stable association of the SmpB.SsrA complex with 70S ribosomes. Using mutational analysis and biochemical experiments, we have discovered a previously uncharacterized SmpB function. This function is required for a step in the tagging process downstream of SsrA binding and ribosome association but before transpeptidation of the SsrA-linked alanine and establishment of the SsrA reading frame. Our results clearly demonstrate that residues in the C-terminal tail of SmpB confer a hitherto unrevealed function that is essential for trans-translation. Based on these results, we propose that upon binding stalled ribosomes, the unstructured C-terminal tail of SmpB acquires contacts that are critical for productive accommodation of SsrA into the ribosomal A site.

Asunto(s)

Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , ARN Bacteriano/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagénesis , Fenotipo , Unión Proteica , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Eliminación de Secuencia

RESUMEN

Simultaneous measurements of membrane capacitance and intracellular calcium concentration were used to examine the calcium dependence of exocytosis in single acinar cells from mouse lacrimal gland and to establish the quantitative relation between calcium concentration and rate of exocytosis. Application of adrenergic or muscarinic agonists elevated intracellular calcium and evoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation was eliminated by internal dialysis with the calcium buffer EGTA, which demonstrated that the increase in intracellular calcium was necessary for agonist-evoked exocytosis. When internal calcium was elevated by application of the calcium ionophore ionomycin, exocytosis was evoked in the absence of agonist stimulation. Thus an increase in intracellular calcium was necessary and sufficient for exocytosis in single acinar cells. The rate of change of membrane capacitance increased as approximately the third power of the calcium concentration, which is similar to the dependence of exocytosis rate on calcium concentration in other secretory cells.

Asunto(s)

Calcio/fisiología , Exocitosis/fisiología , Aparato Lagrimal/metabolismo , Agonistas Adrenérgicos/farmacología , Animales , Calcio/metabolismo , Agonistas Colinérgicos/farmacología , Capacidad Eléctrica , Aparato Lagrimal/citología , Aparato Lagrimal/efectos de los fármacos , Aparato Lagrimal/fisiología , Ratones , Concentración Osmolar