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1.
Biochem Biophys Res Commun ; 443(2): 531-6, 2014 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-24326068

RESUMEN

Ca(2+) signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca(2+) signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca(2+) and pH. Ca(2+) fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca(2+)] increases in human sperm even in the absence of extracellular Ca(2+). Using LysoTracker, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker, suggesting that these stores are the targets of NAADP action.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , NADP/análogos & derivados , Espermatozoides/fisiología , Células Cultivadas , Humanos , Masculino , NADP/metabolismo
2.
Psychol Med ; 44(11): 2409-18, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24467931

RESUMEN

BACKGROUND: Oxidative stress and neurotrophic factors have been implicated in the pathophysiology of bipolar disorder. Our objective was to determine whether plasma glutathione or brain-derived neurotrophic factor (BDNF) levels were abnormal in bipolar disorder and therefore useful as possible biomarkers. METHOD: Blood samples were collected from subsyndromal, medicated bipolar I patients (n = 50), recruited from OXTEXT, University of Oxford, and from 50 matched healthy controls. Total and oxidized glutathione levels were measured using an enzymatic recycling method and used to calculate reduced, percentage oxidized, ratio of reduced:oxidized and redox state. BDNF was measured using an enzyme-linked immunoassay. Self-monitored mood scores for the bipolar group were available (Quick Inventory of Depressive Symptomatology and the Altman Self-Rating Mania Scale) over an 8-week period. RESULTS: Compared with controls, bipolar patients had significantly lower levels of total glutathione and it was more oxidized. BDNF levels were not different. Age of illness onset but not current mood state correlated with total glutathione levels and its oxidation status, so that lower levels of total and reduced glutathione were associated with later onset of disease, not length of illness. CONCLUSIONS: Plasma glutathione levels and redox state detect oxidative stress even in subsyndromal patients with normal BDNF. It may relate to the onset and development of bipolar disorder. Plasma glutathione appears to be a suitable biomarker for detecting underlying oxidative stress and for evaluating the efficacy of antioxidant intervention studies.


Asunto(s)
Trastorno Bipolar/sangre , Factor Neurotrófico Derivado del Encéfalo/sangre , Glutatión/sangre , Estrés Oxidativo/fisiología , Adulto , Edad de Inicio , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo
3.
Trials ; 24(1): 361, 2023 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-37248494

RESUMEN

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code "IB1001-301") for the chronic treatment of symptoms in adult and pediatric patients with NPC. METHODS: This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. DISCUSSION: Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. TRIAL REGISTRATIONS: The trial (IB1001-301) has been registered at www. CLINICALTRIALS: gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10). Registered on 20 December 2021.


Asunto(s)
Enfermedad de Niemann-Pick Tipo C , Humanos , Estudios Cruzados , Leucina/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Calidad de Vida , Método Doble Ciego
4.
Trials ; 22(1): 84, 2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33482890

RESUMEN

BACKGROUND: The lack of approved treatments for the majority of rare diseases is reflective of the unique challenges of orphan drug development. Novel methodologies, including new functionally relevant endpoints, are needed to render the development process more feasible and appropriate for these rare populations and thereby expedite the approval of promising treatments to address patients' high unmet medical need. Here, we describe the development of an innovative master protocol and primary outcome assessment to investigate the modified amino acid N-acetyl-L-leucine (Sponsor Code: IB1001) in three separate, multinational, phase II trials for three ultra-rare, autosomal-recessive, neurodegenerative disorders: Niemann-Pick disease type C (NPC), GM2 gangliosidoses (Tay-Sachs and Sandhoff disease; "GM2"), and ataxia telangiectasia (A-T). METHODS/DESIGN: The innovative IB1001 master protocol and novel CI-CS primary endpoints were developed through a close collaboration between the Industry Sponsor, Key Opinion Leaders, representatives of the Patient Communities, and National Regulatory Authorities. As a result, the open-label, rater-blinded study design is considerate of the practical limitations of recruitment and retention of subjects in these ultra-orphan populations. The novel primary endpoint, the Clinical Impression of Change in Severity© (CI-CS), accommodates the heterogenous clinical presentation of NPC, GM2, and A-T: at screening, the principal investigator appoints for each patient a primary anchor test (either the 8-m walk test (8MWT) or 9-hole peg test of the dominant hand (9HPT-D)) based on his/her unique clinical symptoms. The anchor tests are videoed in a standardized manner at each visit to capture all aspects related to the patient's functional performance. The CI-CS assessment is ultimately performed by independent, blinded raters who compare videos of the primary anchor test from three periods: baseline, the end of treatment, and the end of a post-treatment washout. Blinded to the time point of each video, the raters make an objective comparison scored on a 7-point Likert scale of the change in the severity of the patient's neurological signs and symptoms from video A to video B. To investigate both the symptomatic and disease-modifying effects of treatment, N-acetyl-L-leucine is assessed during two treatment sequences: a 6-week parent study and 1-year extension phase. DISCUSSION: The novel CI-CS assessment, developed through a collaboration of all stakeholders, is advantageous in that it better ensures the primary endpoint is functionally relevant for each patient, is able to capture small but meaningful clinical changes critical to the patients' quality of life (fine-motor skills; gait), and blinds the primary outcome assessment. The results of these three trials will inform whether N-acetyl-L-leucine is an effective treatment for NPC, GM2, and A-T and can also serve as a new therapeutic paradigm for the development of future treatments for other orphan diseases. TRIAL REGISTRATION: The three trials (IB1001-201 for Niemann-Pick disease type C (NPC), IB1001-202 for GM2 gangliosidoses (Tay-Sachs and Sandhoff), IB1001-203 for ataxia telangiectasia (A-T)) have been registered at www.clinicaltrials.gov (NCT03759639; NCT03759665; NCT03759678), www.clinicaltrialsregister.eu (EudraCT: 2018-004331-71; 2018-004406-25; 2018-004407-39), and https://www.germanctr.de (DR KS-ID: DRKS00016567; DRKS00017539; DRKS00020511).


Asunto(s)
Ataxia Telangiectasia , Gangliosidosis GM2 , Enfermedades Neurodegenerativas , Femenino , Humanos , Leucina , Masculino , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Calidad de Vida
5.
Trends Biochem Sci ; 26(8): 482-9, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11504624

RESUMEN

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.


Asunto(s)
Señalización del Calcio/fisiología , NADP/fisiología , NADP/análogos & derivados
6.
Br J Pharmacol ; 175(13): 2599-2610, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29488218

RESUMEN

BACKGROUND AND PURPOSE: Lithium's antidepressant action may be mediated by inhibition of inositol monophosphatase (IMPase), a key enzyme in Gq -protein coupled receptor signalling. Recently, the antioxidant agent ebselen was identified as an IMPase inhibitor. Here, we investigated both ebselen and lithium in models of the 5-HT2A receptor, a Gq -protein coupled receptor involved in lithium's actions. EXPERIMENTAL APPROACH: 5-HT2A receptor function was assessed in mice by measuring the behavioural (head-twitches, ear scratches) and molecular (cortical immediate early gene [IEG] mRNA; Arc, c-fos, Egr2) responses to 5-HT2A receptor agonists. Ebselen and lithium were administered either acutely or repeatedly prior to assessment of 5-HT2A receptor function. Because lithium and 5-HT2A receptor antagonists augment the action of selective serotonin reuptake inhibitors (SSRIs), ebselen was tested for this activity by co-administration with the SSRI citalopram in microdialysis (extracellular 5-HT) experiments. KEY RESULTS: Acute and repeated administration of ebselen inhibited behavioural and IEG responses to the 5-HT2A receptor agonist DOI. Repeated lithium also inhibited DOI-evoked behavioural and IEG responses. In comparison, a selective IMPase inhibitor (L-690330) attenuated the behavioural response to DOI whereas glycogen synthase kinase inhibitor (AR-A014418) did not. Finally, ebselen enhanced the increase in extracellular 5-HT induced by citalopram, and also increased regional brain 5-HT synthesis. CONCLUSIONS AND IMPLICATIONS: Our data demonstrated lithium-mimetic effects of ebselen in different experimental models of 5-HT2A receptor function, probably mediated by IMPase inhibition. This evidence of lithium-like neuropharmacological effects of ebselen adds further support for the clinical testing of ebselen in mood disorders, including as an antidepressant augmenting agent.


Asunto(s)
Antioxidantes/farmacología , Azoles/farmacología , Litio/farmacología , Compuestos de Organoselenio/farmacología , Receptor de Serotonina 5-HT2A/metabolismo , Animales , Antioxidantes/administración & dosificación , Azoles/administración & dosificación , Relación Dosis-Respuesta a Droga , Isoindoles , Litio/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Compuestos de Organoselenio/administración & dosificación
7.
Sci STKE ; 2000(41): pe1, 2000 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-11752598

RESUMEN

This Perspective by Galione and Churchill is one in a series on intracellular calcium release mechanisms. The authors review the evidence for cyclic adenosine diphosphate ribose (cADPR) being a second messenger involved in regulating intracellular calcium. In addition, the physiological stimuli and responses mediated by cADPR are discussed. The Perspective is accompanied by a movie showing a calcium wave triggered by cADPR.


Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/fisiología , Calcio/metabolismo , Sistemas de Mensajero Secundario/fisiología , Animales , Señalización del Calcio/fisiología , ADP-Ribosa Cíclica , Humanos
8.
Cell Calcium ; 32(5-6): 343-54, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12543094

RESUMEN

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles. In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Animales , Calcio/fisiología , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Humanos , Sistemas de Mensajero Secundario/fisiología
9.
Invest Ophthalmol Vis Sci ; 38(5): 855-65, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9112981

RESUMEN

PURPOSE: To identify agonists that elevate cytosolic Ca2+ (Cai2+) in lens cells and to characterize their mechanism of action. METHODS: Digital imaging and the Ca(2+)-reporting dye fura-2 were used to study the effects of agonists and their antagonists of Cai2+ in sheep lens primary cell cultures. RESULTS: Exposing cells to adenosine triphosphate (ATP) and epinephrine increased Cai2+, whereas dopamine, 5-hydroxytryptamine, acetylcholine, histamine, kassinin, bradykinin, and glutamate did not elevate Cai2+. The ATP response was mediated by P2U purinergic receptors based on inhibition by the P2 antagonist suramin and the agonist rank potency order ATP = UTP = ATP gamma S > ADP > AMP > > adenosine; adenine, AMP-CPP, and AMP-PCP were inactive. The epinephrine response was mediated by alpha 1 adrenergic receptors based on the greater potency of the alpha 1 adrenergic selective antagonist prazosin compared to that of the alpha 2 adrenergic selective antagonist yohimbine. More specifically, the epinephrine response was mediated by the alpha 1A adrenergic receptor subtype based on the greater potencies exhibited by the alpha 1A subtype selective competitive antagonists WB 4101 and 5-methylurapidil compared to the alpha 1B and alpha 1D selective antagonists spiperone and BMY 7378, respectively. The agonist-mediated Cai2+ increase was dependent on intracellular Ca2+ stores and was inhibited by the phospholipase C inhibitor U73122. ATP or epinephrine could desensitize the cells to either agonist because of both the depletion of intracellular Ca2+ stores and the downregulation of a common intermediate in the signal transduction pathway. CONCLUSIONS: Ca2+ is mobilized from intracellular stores in the sheep lens by ATP and epinephrine acting through P2U purinergic and the alpha 1A adrenergic receptors, respectively. This confirms previous reports of P2U receptors in lens and provides the first report of alpha 1A adrenergic receptors in the lens.


Asunto(s)
Calcio/metabolismo , Cristalino/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Agonistas Adrenérgicos/farmacología , Antagonistas Adrenérgicos/farmacología , Animales , Células Cultivadas , Citosol/metabolismo , Epinefrina/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Cristalino/citología , Cristalino/efectos de los fármacos , Ovinos , Fosfolipasas de Tipo C/metabolismo
10.
J Biol Chem ; 275(49): 38687-92, 2000 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-11006280

RESUMEN

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.


Asunto(s)
Señalización del Calcio/fisiología , NADP/análogos & derivados , Oocitos/fisiología , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , ADP-Ribosa Cíclica , Difusión , Femenino , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , NADP/farmacocinética , NADP/farmacología , Oocitos/efectos de los fármacos , Erizos de Mar
11.
J Biol Chem ; 276(14): 11223-5, 2001 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-11139579

RESUMEN

Although numerous extracellular stimuli are coupled to increases in intracellular Ca(2+), different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca(2+) increases. We investigated the effect of nicotinic acid adenine dinucleotide phosphate (NAADP) inactivation on spatiotemporal Ca(2+) signals in intact sea urchin eggs. The photorelease of NAADP but not inositol 1,4,5-trisphosphate or cyclic ADP-ribose resulted in self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped that of the second. Specifically, the local release of NAADP created a Ca(2+) gradient that was reversed during the subsequent global release of NAADP. Neither cyclic ADP-ribose nor inositol 1,4,5-trisphosphate showed a similar effect. In contrast to homogenates, NAADP inactivation was reversible in intact eggs with resensitization occurring in approximately 20 min. Because initial NAADP responses affect later responses, NAADP can serve as a mechanism for a Ca(2+) memory that has both spatial and temporal components. This NAADP-mediated Ca(2+) memory provides a novel mechanism for cells to control spatiotemporal Ca(2+) increases.


Asunto(s)
Calcio/metabolismo , NADP/metabolismo , Animales , Transporte Iónico/efectos de los fármacos , NADP/análogos & derivados , NADP/farmacología , Erizos de Mar , Transducción de Señal/efectos de los fármacos
12.
EMBO J ; 20(11): 2666-71, 2001 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-11387201

RESUMEN

In sea urchin eggs, Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self-inactivates but paradoxically induces long-term Ca2+ oscillations. We investigated whether NAADP-induced Ca2+ oscillations arise from the recruitment of other Ca2+ release pathways. NAADP, inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) all mobilized Ca2+ from internal stores but only NAADP consistently induced Ca2+ oscillations. NAADP-induced Ca2+ oscillations were partially inhibited by heparin or 8-amino-cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP3- and cADPR-dependent Ca2+ release. Thapsigargin completely blocked IP3 and cADPR responses as well as NAADP-induced Ca2+ oscillations, but only reduced the NAADP-mediated Ca2+ transient. Following NAADP-mediated release from this Ca2+ pool, the amount of Ca2+ in the Ca2+-induced Ca2+ release stores was increased. These results support a mechanism in which Ca2+ oscillations are initiated by Ca2+ release from NAADP-sensitive Ca2+ stores (pool 1) and perpetuated through cycles of Ca2+ uptake into and release from Ca2+-induced Ca2+ release stores (pool 2). These results provide the first direct evidence in support of a two-pool model for Ca2+ oscillations.


Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Señalización del Calcio/fisiología , Inositol 1,4,5-Trifosfato/metabolismo , NADP/farmacología , Oocitos/metabolismo , Adenosina Difosfato Ribosa/análogos & derivados , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , ADP-Ribosa Cíclica , Femenino , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Modelos Biológicos , NADP/análogos & derivados , Oocitos/efectos de los fármacos , Oscilometría , Receptores Citoplasmáticos y Nucleares/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Erizos de Mar , Tapsigargina/farmacología
13.
J Cell Sci ; 111 ( Pt 9): 1217-25, 1998 May.
Artículo en Inglés | MEDLINE | ID: mdl-9547298

RESUMEN

To further characterize how gap junction-dependent Ca2+ waves propagate between sheep lens cells, we examined the possible roles of inositol 1,4,5-trisphosphate (IP3), Ca2+ and cyclic ADP-ribose (cADPR) in mediating intercellular Ca2+ waves. Second messengers were microinjected into a single cell in a monolayer of sheep lens cells while monitoring cytosolic Ca2+ with fura-2 and fluorescence microscopy. All three compounds initiated intercellular Ca2+ waves, but more cells responded following the injection of either IP3 or cADPR than responded following the injection of Ca2+. When either IP3 or cADPR was co-injected with the Ca2+ chelator EGTA, cytosolic Ca2+ in the injected cell decreased but cytosolic Ca2+ in the adjacent cells increased, indicating that the intercellular messenger was IP3 or cADPR, rather than Ca2+. The phospholipase C inhibitor U73122 eliminated mechanically initiated intercellular Ca2+ waves, indicating that mechanical initiation probably requires IP3 production. In U73122-treated cells, injected IP3 initiated an intercellular Ca2+ wave in which the number of cells responding increased as the amount of IP3 injected increased, indicating that the distance traveled by the Ca2+ wave was dependent on cell-to-cell diffusion of IP3. In contrast, the ability of cADPR both to increase cytosolic Ca2+ in the injected cell and to initiate intercellular Ca2+ waves was greatly attenuated by U73122. In conclusion, Ca2+, IP3 and cADPR can all mediate intercellular Ca2+ waves by passing through gap junction channels, but both IP3 and cADPR are more effective intercellular messengers than Ca2+.


Asunto(s)
Calcio/fisiología , Uniones Comunicantes/fisiología , Inositol 1,4,5-Trifosfato/fisiología , Cristalino/citología , Poli Adenosina Difosfato Ribosa/fisiología , Transducción de Señal/fisiología , Animales , Calcio/farmacología , Comunicación Celular , Células Cultivadas , Quelantes/farmacología , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Transporte Iónico , Cristalino/metabolismo , Microinyecciones , Microscopía Fluorescente , Fosfatidilinositol Diacilglicerol-Liasa , Poli Adenosina Difosfato Ribosa/farmacología , Pirrolidinonas/farmacología , Ovinos , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/fisiología
14.
Am J Physiol ; 276(2): C426-34, 1999 02.
Artículo en Inglés | MEDLINE | ID: mdl-9950770

RESUMEN

Intracellular Ca2+ stores in permeabilized sheep lens cells were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca2+-releasing agents. Inositol 1,4,5-trisphosphate (IP3) or cyclic ADP-ribose (cADPR) released Ca2+ from intracellular Ca2+ stores that were maintained by an ATP-dependent Ca2+ pump. The IP3 antagonist heparin inhibited IP3- but not cADPR-mediated Ca2+ release, whereas the cADPR antagonist 8-amino-cADPR inhibited cADPR- but not IP3-mediated Ca2+ release, indicating that IP3 and cADPR were operating through separate mechanisms. A Ca2+ store sensitive to IP3, cADPR, and thapsigargin appeared to be distributed throughout all intracellular regions. In some cells a Ca2+ store insensitive to IP3, cADPR, thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present in a juxtanuclear region. We conclude that lens cells contain intracellular Ca2+ stores that are sensitive to IP3, cADPR, and thapsigargin, as well as a Ca2+ store that appears insensitive to all these agents.


Asunto(s)
Calcio/metabolismo , Membranas Intracelulares/metabolismo , Cristalino/metabolismo , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Animales , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , ADP-Ribosa Cíclica , Resistencia a Medicamentos , Colorantes Fluorescentes , Fura-2/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacología , Membranas Intracelulares/efectos de los fármacos , Cristalino/citología , Cristalino/efectos de los fármacos , Microscopía Fluorescente , Concentración Osmolar , Ovinos , Especificidad por Sustrato , Tapsigargina/farmacología , Distribución Tisular
15.
J Cell Sci ; 109 ( Pt 2): 355-65, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8838659

RESUMEN

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca(2+)-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2-8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2- to 12-fold from resting levels of approximately 100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanically-stimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+ channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanically-activated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.


Asunto(s)
Calcio/metabolismo , Ojo/metabolismo , Transducción de Señal/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Comunicación Celular , Células Cultivadas , Células Epiteliales , Ojo/citología , Uniones Comunicantes/metabolismo , Estimulación Física , Ovinos
16.
Biochem J ; 352 Pt 3: 725-9, 2000 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-11104679

RESUMEN

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.


Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , NADP/análogos & derivados , NADP/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Extractos Celulares , ADP-Ribosa Cíclica , Concentración de Iones de Hidrógeno , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Ligandos , NADP/antagonistas & inhibidores , Óvulo/citología , Óvulo/metabolismo , Unión Proteica/efectos de los fármacos , Erizos de Mar , Cloruro de Sodio/farmacología
17.
J Biol Chem ; 275(47): 36495-7, 2000 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-11010959

RESUMEN

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing agent in invertebrate eggs that has recently been shown to be active in certain mammalian and plant systems. Little, however, is known concerning the properties of putative NAADP receptors. Here, for the first time, we report binding sites for NAADP in brain. In contrast to sea urchin egg homogenates, [(32)P]NAADP bound reversibly to multiple sites in brain membranes. The rank order of potency of NAADP, 2',3'-cyclic NAADP and 3'-NAADP in displacing [(32)P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca(2+) from homogenates. These data indicate that [(32)P]NAADP likely binds to receptors mediating Ca(2+) mobilization. Autoradiography revealed striking heterogeneity in the distribution of [(32)P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca(2+) signaling in the brain.


Asunto(s)
Calcio/metabolismo , NADP/análogos & derivados , Animales , Sitios de Unión , Células Cultivadas , Masculino , NADP/metabolismo , Ratas , Ratas Sprague-Dawley , Erizos de Mar , Transducción de Señal
18.
Biol Cell ; 92(3-4): 197-204, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11043408

RESUMEN

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.


Asunto(s)
Señalización del Calcio/fisiología , Fertilización/fisiología , NADP/análogos & derivados , NADP/fisiología , Animales , Calcio/metabolismo , NADP/metabolismo , Óvulo/metabolismo , Óvulo/fisiología , Erizos de Mar/metabolismo , Erizos de Mar/fisiología
19.
Am J Physiol Cell Physiol ; 281(3): C972-81, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11502574

RESUMEN

The quantitative effects of Ca(2+) signaling on gap junctional coupling in lens epithelial cells have been determined using either the spread of Mn(2+) that is imaged by its ability to quench the fluorescence of fura 2 or the spread of the fluorescent dye Alexa Fluor 594. Gap junctional coupling was unaffected by a mechanically stimulated cell-to-cell Ca(2+) wave. Furthermore, when cytosolic Ca(2+) concentration (Ca) increased after the addition of the agonist ATP, coupling was unaffected during the period that Ca was maximal. However, coupling decreased transiently approximately 5-10 min after agonist addition when Ca returned to resting levels, indicating that this transient decrease in coupling was unlikely due to a direct action of Ca on gap junctions. An increase in Ca mediated by the ionophore ionomycin that was sustained for several minutes resulted in a more rapid and sustained decrease in coupling (IC(50) ~300 nM Ca(2+), Hill coefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca(2+) waves were too transient to mediate a sustained uncoupling of lens epithelial cells.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Uniones Comunicantes/fisiología , Cristalino/fisiología , Epitelio Pigmentado Ocular/fisiología , Adenosina Trifosfato/farmacología , Animales , Calcio/farmacología , Células Cultivadas , Colorantes Fluorescentes , Fura-2 , Uniones Comunicantes/efectos de los fármacos , Ionomicina/farmacología , Cinética , Manganeso/farmacología , Ovinos , Factores de Tiempo
20.
Nature ; 398(6722): 74-6, 1999 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-10078532

RESUMEN

Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , NADP/análogos & derivados , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/antagonistas & inhibidores , Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/farmacología , Animales , Calcio/agonistas , ADP-Ribosa Cíclica , Heparina/farmacología , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , NADP/antagonistas & inhibidores , NADP/metabolismo , Páncreas/citología , Páncreas/metabolismo , Erizos de Mar
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