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1.
Science ; 294(5544): 1117-22, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11691998

ABSTRACT

SNAREs (soluble NSF-attachment protein receptors) are generally acknowledged as central components of membrane fusion reactions, but their precise function has remained enigmatic. Competing hypotheses suggest roles for SNAREs in mediating the specificity of fusion, catalyzing fusion, or actually executing fusion. We generated knockout mice lacking synaptobrevin/VAMP 2, the vesicular SNARE protein responsible for synaptic vesicle fusion in forebrain synapses, to make use of the exquisite temporal resolution of electrophysiology in measuring fusion. In the absence of synaptobrevin 2, spontaneous synaptic vesicle fusion and fusion induced by hypertonic sucrose were decreased approximately 10-fold, but fast Ca2+-triggered fusion was decreased more than 100-fold. Thus, synaptobrevin 2 may function in catalyzing fusion reactions and stabilizing fusion intermediates but is not absolutely required for synaptic fusion.


Subject(s)
Membrane Fusion , Membrane Proteins/physiology , Synapses/physiology , Synaptic Vesicles/physiology , Vesicular Transport Proteins , Action Potentials , Animals , Calcium/metabolism , Calcium/pharmacology , Cells, Cultured , Hypertonic Solutions , Membrane Proteins/genetics , Mice , Mice, Knockout , Mutation , Patch-Clamp Techniques , Potassium/pharmacology , Presynaptic Terminals/physiology , Prosencephalon/physiology , R-SNARE Proteins , SNARE Proteins , Sucrose/pharmacology , Synaptic Transmission
2.
Trends Neurosci ; 24(11): 637-43, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11672807

ABSTRACT

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.


Subject(s)
Central Nervous System/physiology , Nerve Endings/physiology , Signal Transduction/physiology , Synaptic Vesicles/physiology , Animals
3.
J Cell Biol ; 150(5): 1101-12, 2000 Sep 04.
Article in English | MEDLINE | ID: mdl-10973998

ABSTRACT

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Carrier Proteins/metabolism , Pancreas/cytology , Pancreas/physiology , Actins/physiology , Animals , Brefeldin A/pharmacology , Calcium Channels/isolation & purification , Calcium Channels/physiology , Calcium Signaling/drug effects , Carbachol/pharmacology , Carrier Proteins/analysis , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Polarity , Cells, Cultured , Cytoskeleton/physiology , Cytoskeleton/ultrastructure , GTP-Binding Proteins/isolation & purification , GTP-Binding Proteins/metabolism , Golgi Apparatus/physiology , Golgi Apparatus/ultrastructure , Inositol 1,4,5-Trisphosphate Receptors , Inositol Phosphates/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Proteins , Patch-Clamp Techniques , Rats , Receptors, Cytoplasmic and Nuclear/isolation & purification , Receptors, Cytoplasmic and Nuclear/physiology
4.
Pflugers Arch ; 435(6): 859-64, 1998 May.
Article in English | MEDLINE | ID: mdl-9518516

ABSTRACT

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.


Subject(s)
Calcium/metabolism , Calcium/pharmacology , Neoplasms/metabolism , Uridine Triphosphate/pharmacology , Caffeine/pharmacology , Culture Media , Endothelium/drug effects , Endothelium/metabolism , Humans , Inositol 1,4,5-Trisphosphate/pharmacology , Ryanodine/pharmacology , Thapsigargin/pharmacology , Tumor Cells, Cultured
5.
Pflugers Arch ; 433(1-2): 180-7, 1996.
Article in English | MEDLINE | ID: mdl-9019721

ABSTRACT

Sustained Ca2+ elevation ("Ca2+ response"), caused by subsequent readdition of Ca2+ to the medium after application of adenosine 5'-triphosphate (ATP, 15 microM) in a Ca2+-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca2+ concentration ([Ca2+]i) was between about 50 and 110 nM, a massive Ca2+ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca2+]i of over 110 nM displayed small plateau-like Ca2+ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca2+ response amplitude on resting [Ca2+]i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca2+ responses, the amplitudes of which were almost constant and in the same range as resting [Ca2+]i. Ni2+, known to partly block Ca2+ entry, caused no change in the half-decay time of [Ca2+]i down to the level of the sustained phase [57 +/- 4 s in control and 54 +/- 3 s (n = 13) in the presence of 10 mM Ni2+] when added at the peak of the phasic component of the Ca2+ response. However, it lowered the sustained phase of the Ca2+ response by 42%. When applied at the start of the readdition of Ca2+, Ni2+ blocked the phasic component of the Ca2+ response, there being a threefold decrease in the initial rate of [Ca2+]i rise. In cells with a resting [Ca2+]i of 75-80 nM, pre-treatment with ryanodine (10 microM) did not affect the peak amplitude of the Ca2+ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca2+ response. In conclusion, partial depletion of the inositol 1,4, 5-trisphosphate-(IP3-) sensitive store by a submaximal concentration of agonist (in Ca2+-free medium) was followed, on readdition of Ca2+, by Ca2+ entry, which appeared to trigger IP3-sensitive Ca2+ release (IICR) which, in turn, initiated Ca2+-sensitive Ca2+ release (CICR), thus resulting in a massive elevation of [Ca2+]i.


Subject(s)
Calcium/metabolism , Calcium/pharmacology , Culture Media , Cytosol/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Ryanodine/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Calcium/agonists , Cattle , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Nickel/pharmacology , Osmolar Concentration
6.
Pflugers Arch ; 432(4): 614-22, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8764961

ABSTRACT

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca2+ transients in a Ca2+-free medium have been revealed, which evidently reflects Ca2+ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca2+ ([Ca2+]i) from about 50 to 110 nM, a biphasic dependence of the Ca2+ transients on resting [Ca2+]i was shown and spontaneous Ca2+ oscillations were observed. At a [Ca2+]i level over 110 nM, a pronounced rise in Ca2+ transients occurred and only single transients were observed. Ryanodine (10 microM) produced a transient [Ca2+]i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca2+ release (IICR) and Ca2+-sensitive Ca2+ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca2+ oscillations in BAE cells, whereas the ATP-induced (5-100 microM) Ca2+ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca2+ transients was associated with a [Ca2+]i decrease to a level below the initial resting [Ca2+]i (undershoot). Its depth biphasically depended on the resting [Ca2+]i from 50 to 110 nM, suggesting that the lack of a Ca2+ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca2+ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients.


Subject(s)
Calcium/metabolism , Endothelium, Vascular/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Ryanodine/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Aorta/cytology , Caffeine/pharmacology , Cattle , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects
7.
Gen Physiol Biophys ; 14(5): 427-36, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8786042

ABSTRACT

Contractile responses induced by 25 mumol/l oxytocin in myometrial strips isolated from the uterus of estradiol-dominated rats comprised both phasic and tonic components. In a Ca(2+)-free medium (containing 0.1 mmol/l EGTA and no added Ca2+), the oxytocin-induced contractions seemed to be associated with Ca2+ release from intracellular stores. Frusemide, known to lower the cAMP level in the rat myometrium, did not affect the responses due to Ca2+ release but inhibited those mediated through an acceleration of the Ca2+ influx. The permanent presence of frusemide (1.5 mmol/l) in the CaCl2-containing medium influenced the oxytocin-induced responses in the same manner as did omission of Ca2+ from the medium. The frusemide-sensitive component of the responses to oxytocin was superimposed on a persistent contraction caused by KCl depolarization, suggesting that frusemide completely inhibited the oxytocin-induced Ca2+ influx. At the same time, frusemide moderately (by only 34 +/- 7%) decreased the amplitude of the KCl-induced contracture. This decrease varied with the frusemide concentration, and could be partly prevented by addition of dibutyryl-cAMP; i.e. probably, it was mediated by an inhibition of voltage-gated Ca2+ influx due to a decrease in the intracellular cAMP level. The data presented seem to suggest that in the rat myometrium exposed to oxytocin (25 mumol/l) both voltage-gated and receptor-operated Ca2+ entries are regulated by cAMP-dependent protein kinases.


Subject(s)
Calcium/metabolism , Furosemide/pharmacology , Myometrium/drug effects , Myometrium/metabolism , Oxytocin/pharmacology , Uterine Contraction/drug effects , Uterine Contraction/physiology , Animals , Bucladesine/pharmacology , Calcium Channel Blockers/pharmacology , Cyclic AMP/metabolism , Female , In Vitro Techniques , Intracellular Fluid/metabolism , Ion Transport/drug effects , Rats , Rats, Wistar , Verapamil/pharmacology
8.
Exp Physiol ; 79(5): 661-7, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7818857

ABSTRACT

Oxytocin-induced contractions of isolated strips of oestradiol-treated rat myometrium were found to be affected by exposure to the diuretic frusemide. At a concentration of 20 microM. frusemide transiently increased the force of contraction over a period of approximately 10 min. After this time there was a progressive fall in contractile force. At a higher concentration of 200 microM, only the progressive fall in force was seen until contractions were completely abolished. Frusemide has been reported to increase the activity of cAMP-phosphodiesterase in tissue extracts from oestradiol-treated rat myometrium. Therefore, the changes in contraction due to exposure to frusemide may be a reflection of the changes in intracellular cAMP resulting from a stimulation of cAMP-phosphodiesterase activity. In support of this idea, addition of dibutyryl cAMP was found to partially restore contractions after frusemide treatment. These data suggest that frusemide may be a useful tool in the manipulation of tissue cAMP levels in order to determine the different roles of cAMP in the oestradiol-treated rat myometrium.


Subject(s)
Furosemide/pharmacology , Myometrium/drug effects , Oxytocin/pharmacology , Uterine Contraction/drug effects , Animals , Bucladesine/pharmacology , Cyclic AMP/metabolism , Female , In Vitro Techniques , Myometrium/physiology , Rats , Rats, Wistar
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