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1.
Synaptotagmin (syt) I is thought to act as a Ca2+ sensor that regulates neuronal exocytosis. Fifteen additional isoforms of syt have been identified, but their functions are less well understood. Here, we used PC12 cells to test the idea that different isoforms of syt impart cells with distinct metal (i.e., Ca2+, Ba2+, and Sr2+) requirements for secretion. These cells express syt's I and IX (syt IX sometimes referred to as syt V), which have low apparent metal affinities, at much higher levels than syt VII, which we show has a relatively high apparent affinity for metals. We found that syt I and VII partially colocalize on large dense core vesicles and that upregulation of syt VII produces a concomitant increase in the divalent cation sensitivity of catecholamine release from PC12 cells. Furthermore, RNA interference-mediated knockdown of endogenous syt VII reduced the metal sensitivity of release. These data support the hypothesis that the complement of syt's expressed by a cell, in conjunction with their metal affinity, determines the divalent cation sensitivity of exocytosis.  相似文献   

2.
In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca2+ concentration ([Ca2+]i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca2+]i. Low concentrations of ATP (<10 µM) induced intracellular Ca2+ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca2+]i rise and increased the exocytosis rate sevenfold. The contribution of Ca2+ or cAMP pathways to exocytosis was tested by using the Ca2+ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca2+]i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca2+ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate  相似文献   

3.
In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.  相似文献   

4.
Synaptotagmin (syt) serves as a Ca2+ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca2+, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca2+ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.  相似文献   

5.
Many synaptotagmins are Ca2+-binding membrane proteins with functions in Ca2+-triggered exocytosis. Synaptotagmin IV (syt IV) has no Ca2+ binding activity, but nevertheless modulates exocytosis. Here, cell-attached capacitance recording was used to study single vesicle fusion and fission in control and syt IV overexpressing PC12 cells. Unitary capacitance steps varied widely in size, indicating that both microvesicles (MVs) and dense-core vesicles (DCVs) undergo fusion. Syt IV overexpression reduced the size of DCVs and endocytotic vesicles but not MVs. Syt IV also reduced the basal rate of Ca2+-induced fusion. During kiss-and-run, syt IV increased the conductance and duration of DCV fusion pores but not MV fusion pores. During full-fusion of DCVs syt IV increased the fusion pore conductance but not the duration. Syt IV overexpression increased the duration but not the conductance of fission pores during endocytosis. The effects of syt IV on fusion pores in PC12 cells resembled the effects on fusion pores in peptidergic nerve terminals. However, differences between these and results obtained with amperometry may indicate that amperometry and capacitance detect the fusion of different populations of vesicles. The effects of syt IV on fusion pores are discussed in terms of structural models and kinetic mechanisms.  相似文献   

6.
Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect mean cellular Q or the proportional release from different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free defined medium. After two extra days of culture in the defined medium, there was a rundown in mean cellular Q, and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplementation but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca2+ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca2+ concentration, we found that for cells cultured in defined medium dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting the VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca2+. Thus glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Because chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the colocalization of releasable granules with VGCCs. catecholamines; paracrine action; exocytosis; calcium channels  相似文献   

7.
From video imaging of fura 2-loaded baby hamster kidney (BHK)cells stably expressing the cloned human glucagon receptor, we foundthe Ca2+ response to glucagon tobe specific, dose dependent, synchronous, sensitive to pertussis toxin,and independent of Ca2+ influx.Forskolin did not elicit a Ca2+response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3',5'-cyclicmonophosphothioate, resulted in a reduced glucagon-mediatedCa2+ response as well asCa2+ oscillations. The specificphospholipase C inhibitor U-73122 abolished theCa2+ response to glucagon, and amodest twofold increase in inositol trisphosphate(IP3) production could beobserved after stimulation with glucagon. In BHK cells coexpressingglucagon and muscarinic (M1)acetylcholine receptors, carbachol blocked the rise in intracellular free Ca2+ concentrations inresponse to glucagon, whereas glucagon did not affect thecarbachol-induced increase inCa2+. Furthermore, carbachol, butnot glucagon, could block thapsigargin-activated increases inintracellular free Ca2+concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation ofphospholipase C and the release ofCa2+ fromIP3-sensitive intracellularCa2+ stores. Finally, we provideevidence to suggest that cAMP potentiates theIP3-mediated effects onintracellular Ca2+ handling.

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8.
Antisense oligodeoxynucleotides (AS-oligos) targeted to theNa+/Ca2+exchanger (NCX) inhibit NCX-mediatedCa2+ influx in mesenteric artery(MA) myocytes [Am. J. Physiol.269 (Cell Physiol. 38):C1340-C1345, 1995]. Here, we show AS-oligo knockdown ofNCX-mediated Ca2+ efflux. Ininitial experiments, the cytosolic freeCa2+ concentration([Ca2+]cyt)was raised, and sarcoplasmic reticulum (SR)Ca2+ sequestration was blockedwith caffeine and cyclopiazonic acid; the extracellularNa+-dependent (NCX) component ofCa2+ efflux was then selectivelyinhibited in AS-oligo-treated cells but not in controls (no oligos ornonsense oligos). In contrast, theLa3+-sensitive (plasmalemmaCa2+ pump) component ofCa2+ efflux was unaffected inAS-oligo-treated cells. Knockdown of NCX activity was reversed byincubating AS-oligo-treated cells in normal media for 5 days. Transient[Ca2+]cytelevations evoked by serotonin (5-HT) at 15-min intervals inAS-oligo-treated cells were indistinguishable from those in controls.When cells were stimulated every 3 min, however, the peak amplitudes ofthe second and third responses were larger, and[Ca2+]cytreturned to baseline more slowly, in AS-oligo-treated cells than incontrols. Peak 5-HT-evoked responses in the controls, but notAS-oligo-treated cells, were augmented more than twofold inNa+-free media. This implies thatNCX is involved in Na+ gradientmodulation of SR Ca2+ stores andcell responsiveness. The repetitive stimulation data suggest that theNCX may be important during tonic activation of arterial myocytes.

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9.
To investigate thepossible role of members of the mammalian transient receptor potential(TRP) channel family (TRPC1-7) in vasoconstrictor-inducedCa2+ entry in vascular smooth muscle cells, we studied[Arg8]-vasopressin (AVP)-activated channels in A7r5aortic smooth muscle cells. AVP induced an increase in free cytosolicCa2+ concentration ([Ca2+]i)consisting of Ca2+ release and Ca2+ influx.Whole cell recordings revealed the activation of a nonselective cationcurrent with a doubly rectifying current-voltage relation strikinglysimilar to those described for some heterologously expressed TRPCisoforms. The current was also stimulated by direct activation of Gproteins as well as by activation of the phospholipase C-coupledplatelet-derived growth factor receptor. Currents were not activated bystore depletion or increased [Ca2+]i.Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property ofthe TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currentsin A7r5 cells were increased by flufenamate. Northern hybridizationrevealed mRNA coding for TRPC1 and TRPC6. We therefore suggest thatTRPC6 is a molecular component of receptor-stimulated Ca2+-permeable cation channels in A7r5 smooth muscle cells.

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10.
Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca2+ concentration([Ca2+]i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm2). Before application of shear, cells were treatedwith two Ca2+ channel inhibitors or various blockers ofintracellular Ca2+ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca2+]i response, neithergadolinium nor nifedipine, an L-type channel Ca2+ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca2+ chelator, or thapsigargin,which empties intracellular Ca2+ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP3-mediated intracellular Ca2+release is required for modulating flow-induced responses in MC3T3-E1 cells.

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11.
Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca2+ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca2+ concentration ([Ca2+]cyt) were observed in the absence of extracellular Ca2+. Capacitative Ca2+ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca2+ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca2+ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca2+ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca2+-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca2+]cyt. In PAEC bathed in Ca2+-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca2+]cyt but did not affect initial transient increase in [Ca2+]cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca2+]cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca2+ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca2+ release. intracellular calcium stores; oscillations; pulmonary hypertension  相似文献   

12.
Synaptotagmin VII (Syt VII), which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.  相似文献   

13.
Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca2+, although the level of translatable-amylase mRNA was not affected in the presence of Ca2+. Sugardepletion markedly stimulated Ca2+ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca2+ and that of -amylasemolecules induced in the sugar-depleted cells. Ca2+ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa2+ channel blocker, La3+ significantly inhibited the Ca2+uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca2+ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. 4 Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.  相似文献   

14.
Synaptotagmin I (Syt I),a low-affinity Ca2+-binding protein, is thought to serve asthe Ca2+ sensor in the release of neurotransmitter.However, functional studies on the calyx of Held synapse revealed thatthe rapid release of neurotransmitter requires only approximatelymicromolar [Ca2+], suggesting that Syt I may play a morecomplex role in determining the high-affinity Ca2+dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinityCa2+-dependent exocytic pathways and express Syt I. Usingpatch-clamp capacitance measurements to monitor secretion and the acuteantisense deletion of Syt I from differentiated cells, we have shownthat the rapid and the most Ca2+-sensitive pathway ofexocytosis in rat melanotrophs requires Syt I. Furthermore, stimulationof the Ca2+-dependent exocytosis by cytosol dialysis withsolutions containing 1 µM [Ca2+] was completelyabolished in the absence of Syt I. Similar results were obtained by thepreinjection of antibodies against the CAPS (Ca2+-dependentactivator protein for secretion) protein. These results indicate thatsynaptotagmin I and CAPS proteins increase the probability of vesiclefusion at low cytosolic [Ca2+].

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15.
In thetilapia (Oreochromis mossambicus), as in many euryhalineteleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and waterpermeability and increase solute retention. Consistent with theseactions, PRL release is stimulated as extracellular osmolality isreduced both in vivo and in vitro. In the current experiments, aperfusion system utilizing dispersed PRL cells was developed forpermitting the simultaneous measurement of cell volume and PRL release.Intracellular Ca2+ was monitored using fura 2-loaded cellsunder the same conditions. When PRL cells were exposed to hyposmoticmedium, an increase in PRL cell volume preceded the increase in PRLrelease. Cell volume increased in proportion to decreases of 15 and30% in osmolality. However, regulatory volume decrease was clearlyseen only after a 30% reduction. The hyposmotically induced PRLrelease was sharply reduced in Ca2+-deleted hyposmoticmedium, although cell volume changes were identical to those observedin normal hyposmotic medium. In most cells, a rise in intracellularCa2+ concentration ([Ca2+]i)during hyposmotic stimulation was dependent on the availability ofextracellular Ca2+, although small transient increases in[Ca2+]i were sometimes observed uponintroduction of Ca2+-deleted media of the same or reducedosmolality. These results indicate that an increase in cell size is acritical step in the transduction of an osmotic signal into PRL releaseand that the hyposmotically induced increase in PRL release is greatlydependent on extracellular Ca2+.

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16.
This study examines theCa2+ influx-dependent regulationof the Ca2+-activatedK+ channel(KCa) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theKCa current and cytosolic Ca2+ concentration([Ca2+]i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa2+ and addition ofLa3+ orGd3+, but notZn2+, inhibited the increases inKCa current and[Ca2+]i.Ca2+ influx during refill (i.e.,addition of Ca2+ to cells treatedwith CCh and then atropine inCa2+-free medium) failed to evokeincreases in the KCa current but achieved internal Ca2+ storerefill. When refill was prevented by thapsigargin,Ca2+ readdition induced rapidactivation of KCa. These dataprovide further evidence that intracellularCa2+ accumulation provides tightbuffering of[Ca2+]iat the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca2+ influx-dependentregulation of the sustained KCacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa2+ into the internalCa2+ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

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17.
We previously reported that human growth hormone (hGH) increases cytoplasmic Ca2+ concentration ([Ca2+]i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca2+]i involves Ca2+-induced Ca2+ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca2+]i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca2+]i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K+ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca2+]i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K+. Ovine prolactin increased [Ca2+]i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca2+]i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca2+]i. Our study indicates that hGH stimulates rise in [Ca2+]i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca2+-induced Ca2+ release  相似文献   

18.
IntracellularCa2+ release channels such asryanodine receptors play crucial roles in theCa2+-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa2+ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa2+ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca2+ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca2+ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa2+ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.

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19.
Mucin secretion by airway goblet cells is under the control ofapical P2Y2, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca2+ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa2+ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa2+ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa2+EC50 was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa2+ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa2+ activities tested: at 10 nMCa2+ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca2+ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca2+. Hence, SPOC1cells possess Ca2+-insensitive,PKC-dependent, and Ca2+-dependentPKC-potentiated pathways for mucin granule exocytosis.

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20.
To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca2+ imaging  相似文献   

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