共查询到20条相似文献,搜索用时 15 毫秒
1.
Seale AP Richman NH Hirano T Cooke I Grau EG 《American journal of physiology. Cell physiology》2003,284(5):C1280-C1289
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+. 相似文献
2.
Yano S Ishikawa T Tsuda H Obara K Nakayama K 《American journal of physiology. Cell physiology》2005,288(3):C702-C709
A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca2+ and by the voltage-dependent Ca2+ channel (VDCC) blocker nicardipine, but it was unaffected by thapsigargin, which depletes intracellular Ca2+ stores. The contraction was also inhibited by Gd3+ and ruthenium red, cation channel blockers, and Cl channel blockers DIDS and niflumic acid. The reduction of extracellular Cl concentrations enhanced the hypotonically induced contraction. Patch-clamp analysis showed that a hyposmotic challenge activated outwardly rectifying whole cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl concentration, indicating that the currents were carried by Cl. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca2+. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca2+ influx, thereby activating Ca2+-activated Cl channels. The subsequent membrane depolarization is likely to increase Ca2+ influx through VDCC and elicit contraction. stretch-activated cation channels; Ca2+-activated Cl channels; voltage-dependent Ca2+ channels; large-conductance Ca2+-activated K+ channels; gadolinium 相似文献
3.
4.
Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin 总被引:1,自引:0,他引:1
Mongin Alexander A.; Cai Zhaohui; Kimelberg Harold K. 《American journal of physiology. Cell physiology》1999,277(4):C823
Cell swelling results in regulatory activation of multipleconductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement ofextracellular and intracellularCa2+ in volume-dependent[3H]taurine effluxfrom primary cultured astrocytes and compare theCa2+ sensitivity of this efflux inslow (high K+ medium induced) andfast (hyposmotic medium induced) cell swelling. NeitherCa2+-free medium norCa2+-channel blockers prevented thevolume-dependent[3H]taurine release.In contrast, loading cells with the membrane-permeable Ca2+ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed[3H]taurine efflux by65-70% and 25-30% underhigh-K+ and hyposmotic conditions,respectively. Fura 2 measurements confirmed that BAPTA-AM, but notCa2+-free media, significantlyreduced resting intracellular Ca2+concentration([Ca2+]i).The calmodulin antagonists trifluoperazine and fluphenazine reversiblyand irreversibly, respectively, inhibited thehigh-K+-induced[3H]taurine release,consistent with their known actions on calmodulin. In hyposmoticconditions, the effects were less pronounced. These data suggest thatvolume-dependent taurine release requires minimal basal[Ca2+]iand involves calmodulin-dependent step(s). Quantitative differences inCa2+/calmodulin sensitivity ofhigh-K+-induced and hyposmoticmedium-induced taurine efflux are due to both the effects of theinhibitors on high-K+-induced cellswelling and their effects on transport systems and/or signalingmechanisms determining taurine efflux. 相似文献
5.
Bull R Finkelstein JP Humeres A Behrens MI Hidalgo C 《American journal of physiology. Cell physiology》2007,293(1):C162-C171
Despite their relevance for neuronal Ca2+-induced Ca2+ release (CICR), activation by Ca2+ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg2+], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg2+], and RyR redox state on the Ca2+ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg2+] up to 1 mM inhibited vesicular [3H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg2+ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca2+ dependencies, defined by low, moderate, or high maximal fractional open time (Po), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca2+] for activation, increased maximal Po, and shifted channel inhibition to higher [Ca2+]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg2+] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg2+] induced a right shift in Ca2+ dependence for all channels so that [Ca2+] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca2+ at physiological [ATP] and [Mg2+]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca2+-induced Ca2+ release; Ca2+ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor 相似文献
6.
Lee SY Choi BH Hur EM Lee JH Lee SJ Lee CO Kim KT 《American journal of physiology. Cell physiology》2006,290(4):C1060-C1066
Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca2+ influx is triggered by NE, the Ca2+ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca2+ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca2+ response after repetitive stimulation. Ca2+ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca2+ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca2+ entry. Moreover, NE failed to induce further Ca2+ entry after the development of Ca2+ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca2+ entry induced by NE might be identical to that of Tg. Interestingly, Ca2+ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca2+-activated K+ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca2+ entry coupled to BK channels and that NE-induced Ca2+ entry is important in the regulation of AANAT. serotonin N-acetyltransferase; pineal gland 相似文献
7.
Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells 总被引:1,自引:0,他引:1
Ben-Tabou De-Leon S Ben-Zeev G Nussinovitch I 《American journal of physiology. Cell physiology》2006,290(1):C222-C232
Increased extracellular osmolarity ([Os]e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca2+ influx may mediate this effect. We show that increase in [Os]e (by 18125%) differentially suppresses L-type and T-type Ca2+ channel currents (IL and IT, respectively); IL was more sensitive than IT. Hyperosmotic suppression of IL depended on the magnitude of increase in [Os]e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of IL and IT persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca2+ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca2+ influx involves Ca2+ channel proteins. We therefore recorded the activity of L-type Ca2+ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]e reduced the activity of Ca2+ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca2+ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca2+ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity 相似文献
8.
Ding Y Schwartz D Posner P Zhong J 《American journal of physiology. Cell physiology》2004,287(2):C413-C421
It has been suggested that L-type Ca2+ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba2+ currents (IBa) through L-type Ca2+ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel activity but did not alter the voltage-dependent characteristics of Ca2+ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-318425 or Go-6983, prevented IBa enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased IBa under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on IBa when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current 相似文献
9.
Activation of calcium release assessed by calcium release-induced inactivation of calcium current in rat cardiac myocytes 总被引:4,自引:0,他引:4
Zahradníková A Kubalová Z Pavelková J Györke S Zahradník I 《American journal of physiology. Cell physiology》2004,286(2):C330-C341
In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current (ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from 50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus. excitation-contraction coupling 相似文献
10.
Lee S Kim JC Li Y Son MJ Woo SH 《American journal of physiology. Cell physiology》2008,294(4):C966-C976
This study examines whether fluid pressure (FP) modulates the L-type Ca2+ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca2+ current (ICa) and cytosolic Ca2+ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed ICa (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of ICa. The level of ICa suppression by FP depended on the level and duration of pressure. The Ba2+ current through the Ca2+ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca2+ channel during FP stimulation. The cytosolic Ca2+ transients and the basal Ca2+ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the ICa and on the Ca2+ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca2+ buffer, eliminated the FP-induced acceleration of ICa inactivation and reduced the inhibitory effect of the FP on ICa by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca2+ release, eliminated the accelerating effect of FP on the ICa inactivation, and they reduced the inhibitory effect of FP on the ICa. These results suggest that the fluid pressure indirectly suppresses the Ca2+ channel by enhancing the Ca2+-induced intracellular Ca2+ release in rat ventricular myocytes. L-type Ca2+ current; fluid pressure; ventricular myocytes; cytosolic Ca2+ transient 相似文献
11.
An internodal cell of Nitella flexilis treated with 10 mM KC1was vacuole-perfused with an isotonic solution containing ethyleneglycol-bis-(rß-aminoethylether)N,N,N,N-tetraaceticacid (EGTA) and its content including cytoplasm was squeezedout into a vessel and covered with silicone oil. When the hypotonicsolution was added into the cytoplasmic drop which had beenmixed with aequorin, a significant increase in the light emissionfrom aequorin was detected with the photomultiplier, indicatinga release of Ca2+ from some cell organdies storing Ca2+. Thisincrease in the light emission was strongly inhibited by treatingcells with 0.1 mM HgCl2 which is known to inhibit water channelsin the plasma membrane. The inhibition was completely recoveredby washing HgCl2 with 2-mer-captoethanol. This suggests thatwater channels may exist in the membrane of Ca2+ stores andplay an essential role in the hydration-induced Ca2+ release. (Received February 12, 1998; Accepted May 21, 1998) 相似文献
12.
Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca2+-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca2+ from internal stores,since influx of Ca2+ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa2+ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca2+ isconcluded to be caused by the release of Ca2+ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca2+ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994) 相似文献
13.
Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation 总被引:16,自引:0,他引:16
Platoshyn O Golovina VA Bailey CL Limsuwan A Krick S Juhaszova M Seiden JE Rubin LJ Yuan JX 《American journal of physiology. Cell physiology》2000,279(5):C1540-C1549
Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca2+ ([Ca2+]cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (Em) regulates[Ca2+]cyt by governing Ca2+influx through voltage-dependent Ca2+ channels. Thusintracellular Ca2+ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K+(Kv) channels regulates resting Em. In thisstudy, we investigated whether changes of Kv currents[IK(V)], Em, and[Ca2+]cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca2+ abolished PASMC growth. Resting[Ca2+]cyt was significantly higher, andresting Em was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell IK(V) was significantly attenuated in PASMCduring proliferation. Furthermore, Emdepolarization significantly increased resting[Ca2+]cyt and augmented agonist-mediatedrises in [Ca2+]cyt in the absence ofextracellular Ca2+. These results demonstrate that reducedIK(V), depolarized Em, and elevated [Ca2+]cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca2+]cyt in PASMC. 相似文献
14.
Mauban JR Wilkinson K Schach C Yuan JX 《American journal of physiology. Cell physiology》2006,290(2):C325-C336
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; 1530 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
15.
Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration 总被引:2,自引:0,他引:2
Wang JY Wang J Golovina VA Li L Platoshyn O Yuan JX 《American journal of physiology. Cell physiology》2000,278(2):C303-C314
Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K+ channels (Kv) controlsmembrane potential (Em) that regulates cytoplasmicfree Ca2+ concentration([Ca2+]cyt) by governing thedriving force for Ca2+ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K+ channel gene expression,Em, and[Ca2+]cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca2+ channels, the depolarizedEm in DFMO-treated cells decreased [Ca2+]cyt as a result of reduceddriving force for Ca2+ influx through capacitativeCa2+ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, Em,and [Ca2+]cyt but also restoredcell migration to normal. Removal of extracellular Ca2+ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca2+]cyt by increasing the drivingforce (the electrochemical gradient) for Ca2+ influx andthus stimulates cell migration. 相似文献
16.
A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle 总被引:3,自引:0,他引:3
Zhou J Yi J Royer L Launikonis BS González A García J Ríos E 《American journal of physiology. Cell physiology》2006,290(2):C539-C553
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
17.
Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C 总被引:1,自引:0,他引:1
Bayguinov O Hagen B Kenyon JL Sanders KM 《American journal of physiology. Cell physiology》2001,281(5):C1512-C1523
Localized Ca2+ transients resulting from inositoltrisphosphate (IP3)-dependent Ca2+ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa2+ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca2+([Ca2+]o) reduced localized Ca2+transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increasedcoupling strength between Ca2+ transients and STOCs when[Ca2+]o was reduced. Gd3+ (10 µM) did not affect Ca2+ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca2+influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca2+ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca2+ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca2+ transients. Single channelstudies indicated that large-conductance Ca2+-activatedK+ (BK) channels were inhibited by aCa2+-dependent PKC. In summary 1)Ca2+ release from IP3 receptor-operated storesactivates Ca2+-activated K+ channels;2) Ca2+ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca2+ sensitivity of BK channels, reducing the couplingstrength between localized Ca2+ transients and BK channels. 相似文献
18.
Denson Donald D.; Worrell Roger T.; Middleton Pamela; Eaton Douglas C. 《American journal of physiology. Cell physiology》1999,276(1):C201
To test thehypothesis that intracellular Ca2+activation of large-conductanceCa2+-activatedK+ (BK) channels involves thecytosolic form of phospholipase A2 (cPLA2), we first inhibited theexpression of cPLA2 by treating GH3 cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA2. Western blot analysis and a biochemical assay of cPLA2activity showed marked inhibition of the expression ofcPLA2 in antisense-treated cells.We then examined the effects of intracellularCa2+ concentration([Ca2+]i)on single BK channels from these cells. Open channel probability (Po) for thecells exposed to cPLA2 antisenseoligonucleotides in 0.1 µM intracellularCa2+ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of thePo-voltagerelationship). In fact, a 1,000-fold increase in[Ca2+]ifrom 0.1 to 100 µM did not significantly increasePoin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalPo-[Ca2+]iresponse. Finally, we examined the effect of exogenous arachidonic acidon thePoof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increasePo,it did so without restoring the[Ca2+]isensitivity observed in untreated cells. We conclude that although [Ca2+]idoes impart some basal activity to BK channels inGH3 cells, the steepPo-[Ca2+]irelationship that is characteristic of these channels involves cPLA2. 相似文献
19.
Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores 总被引:4,自引:0,他引:4
Martin Cecile; Hyvelin Jean-Marc; Chapman Karen E.; Marthan Roger; Ashley Richard H.; Savineau Jean-Pierre 《American journal of physiology. Cell physiology》1999,277(2):C243
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. 相似文献
20.
Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding 总被引:5,自引:0,他引:5
Rao JN Platoshyn O Li L Guo X Golovina VA Yuan JX Wang JY 《American journal of physiology. Cell physiology》2002,282(4):C885-C898
Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K+ (Kv) channel expression andcytosolic free Ca2+ concentration([Ca2+]cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca2+ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K+ currents, membrane hyperpolarization, and arise in [Ca2+]cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca2+]cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca2+]cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca2+ influx during restitution. 相似文献