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1.
Kinetic Characterization of Two Phosphate Uptake Systems with Different Affinities in Suspension-Cultured Catharanthus roseus Protoplasts 总被引:2,自引:0,他引:2
We studied the kinetics of inorganic phosphate (P1) uptake from0.11,000 µM P1 by protoplasts from suspension-culturedcells of Catharanthus roseus (L.) G. Don. Concentration dependenceof [32P]P1 uptake revealed two kinetically different uptakesystems, a high-affinity system and a low-affinity system, withKm values of 3.0 and 47 µM, respectively. Protoplastsfrom cells grown in Pi-rich media had a medium level of thelow-affinity activity and a very low level of the high-affinityactivity. It appeared low-affinity system is expressed constitutively,while the high-affinity system is regulated by the availabilityof Pi. When cells grown in a Pi-rich media were transferredto Pi-depleted media, the high-affinity activity increased significantlyafter 2 d, but the low-affinity activity was barely changed.Upon addition of 10 mM Pi, the high level of the high-affinityactivity fell to almost undetectable level in 1d. Both uptakesystems exhibited maximum activity between pH 5 and 6.
1 Present address: Tokyo Research Laboratories, Kyowa HakkoKogyo Co., Ltd., 3-6-6 Asahi-cho, Machida, Tokyo, 194 Japan. 相似文献
2.
Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2 总被引:1,自引:0,他引:1
Bøttger P Hede SE Grunnet M Høyer B Klaerke DA Pedersen L 《American journal of physiology. Cell physiology》2006,291(6):C1377-C1387
The general phosphate need in mammalian cells is accommodated by members of the Pi transport (PiT) family (SLC20), which use either Na+ or H+ to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi (NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with 32Pi as a traceable Pi source. For PiT1, the Michaelis-Menten constant for Pi was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in Pi uptake with a half-maximal activity constant for Pi of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na+-dependent Pi uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+ dependency patterns. However, only PiT2 was capable of Na+-independent Pi transport at acidic pH. Study of the impact of divalent cations Ca2+ and Mg2+ revealed that Ca2+ was important, but not critical, for NaPi transport function of PiT proteins. To gain insight into the NaPi cotransport function, we analyzed PiT2 and a PiT2 Pi transport knockout mutant using 22Na+ as a traceable Na+ source. Na+ was transported by PiT2 even without Pi in the uptake medium and also when Pi transport function was knocked out. This is the first time decoupling of Pi from Na+ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55 and E575 are responsible for linking Pi import to Na+ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20 相似文献
3.
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
4.
To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P11 translocator(s)in isolated chloroplasts from the C3 and CAM-induced plants.The [14C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C3-mode, while a similar high rate of [32P]Pi uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high Vmax [10.6µmol (mg Chl)1 h1] and a comparatively lowKm value (0.41 mM); the latter was similar to Ki values of Pi,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha Ki value (8.4 mM) 20-fold higher than the Km value for G6Puptake, while that in C3 chloroplasts was not inhibited at all.These results suggest that a new G6P/Pi, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C3-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997) 相似文献
5.
Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle 总被引:1,自引:0,他引:1
Shtifman A Paolini C López JR Allen PD Protasi F 《American journal of physiology. Cell physiology》2004,287(1):C73-C78
Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (Pi) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of Pi into the cell must equal the rate of efflux, which is expected to be a function of intracellular Pi concentration. We measured 32P-labeled Pi uptake rates in different muscle fiber types to determine whether they are proportional to cellular Pi content. Pi uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g1·h1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g1·h1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g1·h1). Reasonably similar rates were obtained in vivo. Pi uptake rates at plasma Pi concentrations of 0.31.7 mM confirm that the Pi uptake process is nearly saturated at normal plasma Pi levels. Pi uptake rate correlated with cellular Pi content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of Pi uptake across fiber types implies that other factors are involved in regulating Pi uptake in skeletal muscle. Furthermore, fractional turnover of the cellular Pi pool (0.67, 0.57, and 0.33 h1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular Pi, likely due to differences in resistance to Pi efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux 相似文献
6.
Bull R Marengo JJ Finkelstein JP Behrens MI Alvarez O 《American journal of physiology. Cell physiology》2003,285(1):C119-C128
We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 12631277, 1998), with low, moderate, and high maximal fractional open times (Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 µM cytoplasmic [Ca2+]. At 10 µM [Ca2+], low-Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (KaATP) = 422 µM] than moderate-Po channels (KaATP = 82 µM). Oxidation of low-Po channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-Po channels and decreased KaATP from 422 to 82 µM. At 0.1 µM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low-Po channels. After oxidation to high-Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-Po channels at 10 µM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode. subconductance states; calcium ion release channels; calcium ion regulation; thimerosal; 2,2'-dithiodipyridine 相似文献
7.
L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis 总被引:2,自引:0,他引:2
We investigatedthe role of intracellular calcium concentration([Ca2+]i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca2+]i, and the presence of L-typevoltage-dependent Ca2+ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca2+-chelatingagent EDTA (100 mM), the L-type Ca2+ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca2+channels in endothelial cells, the [Ca2+]isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca2+]i. Pretreatment ofendothelial cells with high K+ (60 mM) or nifedipine (4 µM) diminished increases in [Ca2+]i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca2+]i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca2+]i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca2+ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca2+ channels. 相似文献
8.
Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO2 in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39
[EC]
) that is caused by a decreasein the level of Pi in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by Pi was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of Pi, in a CO2-free preincubationmedium in the presence of 5 mM MgCl2 The maximum value [6.3µmole CO2 (mg Chl)1 min1] was obtained atapproximately 5 mM Pi after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO2 and Mg2+ ions by the gel filtration was 5.29.3nmole CO2 (mg protein)1 min1 and was also enhancedby Pi plus Mg2+ ions. The maximum value [147151 nmoleCO2 (mg protein)1 min1] was attained at 5 mM Piafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof Pi to the enzyme and removal of Pi from the enzyme. Levelsof free Pi and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free Pi in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992) 相似文献
9.
The effect of Na+ on phosphate uptake was studied in four strainsof cyanobacteria: Synechococcus PCC 7942, Gloeothece PCC 6501,Phormidium sp. and Chlorogloeopsis PCC 6912. Phosphate uptakewas stimulated by Na+ in all cases. Li+ and K+ acted as partialanalogues for Na+. Half-saturation [K1/2(Na+)] of phosphateuptake was reached with Na+ concentrations ranging from 317µM in Chlorogloeopsis to 659 µM in Phormidium. Theconcentration of phosphate required to reach half-saturationof phosphate uptake [K1/2(Pi)]was not changed by the presenceof Na+. (Received April 11, 1994; Accepted July 5, 1994) 相似文献
10.
Three marine phytoplankton species (Skeletonema costatum, Olisthodiscusluteus andGonyaulax tamarensis) were grown in batch culturesat 15°C and a 14:10 L:D cycle at irradiance levels rangingfrom 5 to 450 µEinst m2 s1. At each irradiance,during exponential growth, concurrent measurements were madeof cell division, carbon-specific growth rate, photosyntheticperformance (both O2 and POC production), dark respiration,and cellular composition in terms of C, N and chlorophyll a.The results indicate that the three species were similar withrespect to chemical composition, C:N (atomic) = 6.9 ±0.4, photo-synthetic quotient, 1.43 ± 0.09, and photosyntheticefficiency, 2.3 ±0.1 x 103 µmol O2 (µgChl a)1 h1 (µEinst m2 s1)1.Differences in maximum growth rate varied as the 0.24power of cell carbon. Differences in growth efficiency, werebest explained by a power function of Chl a:C at µ = 0.Compensation intensities, ranged from 1.1 µEinst m2s1 for S. costatum to 35 forG. tamarensis and were foundto be a linear function of the maintenance respiration rate.The results indicate that interspecific differences in the µIrelationship can be adequately explained in terms of just threeparameters: cell carbon at maximum growth rate, the C:Chl aratio (at the limit as growth approaches zero) and the respirationrate at zero growth rate. A light-limited algal growth modelbased on these results gave an excellent fit to the experimentalµI curves and explained 97% of the observed interspecificvariability.
1Present address: Lamont-Doherty Geological Observatory Columbiaof University, Palisades, NY 10964, USA 相似文献
11.
Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart 总被引:3,自引:0,他引:3
Poderoso Juan J.; Peralta Jorge G.; Lisdero Constanza L.; Carreras Maria Cecilia; Radisic Marcelo; Schopfer Francisco; Cadenas Enrique; Boveris Alberto 《American journal of physiology. Cell physiology》1998,274(1):C112
Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O2uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O2 · min1 · gtissue1 (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H2O2) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O2 uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O2uptake and H2O2 release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O2uptake and superoxide anion production (reflected byH2O2), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation. 相似文献
12.
Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception 总被引:1,自引:0,他引:1
Vicario I Obeso A Rocher A López-Lopez JR González C 《American journal of physiology. Cell physiology》2000,279(1):C51-C61
Thenotion that intracellular Ca2+ (Cai2+)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa2+ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Cai2+ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca2+ concentration([Ca2+]i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca2+. We found thatthreshold [Ca2+]i for high extracellularK+ (Ke+) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high Ke+)-inducedrelease of CA. The same agents produced Cai2+transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Cai2+responses elicited by high Ke+. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca2+]i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Cai2+ stores do not playa significant role in the instant-to-instant chemoreception process. 相似文献
13.
Sawada Shinichi; Usuda Hideaki; Hasegawa Yasuhide; Tsukui Takahiro 《Plant & cell physiology》1990,31(5):697-704
The results of our previous study [Sawada et al. (1989) PlantCell Physiol. 30: 691] implied that, under sink-limited conditions,a decrease in the activity of ribulose-l,5-bisphosphate carboxylase(EC 4.1.1.39
[EC]
) caused a reduction in the rate of photosyntheticfixation of CO2 in single-rooted leaves of soybean (Glycinemax L. Merr. cv. Tsurunoko). This reduction in the rate of photosynthesisin source leaves seemed to correspond to a decrease in the demandby sink tissues for photoassimilates. In the present study,the activity of RuBPcase in vivo was estimated by measuringthe "initial" activity immediately after extraction from standardleaves (grown under a regime of 10 h of light and 14 h of darkness)and from sink-limited leaves (exposed for 6 or 7 d to continuouslight to alter the source/sink balance). The rate of photosynthesisin the sink-limited leaves decreased to 47% of that in the standardleaves. The "initial" activity of RuBPcase was 4.3 in the standardleaves but only 1.6 µmole CO2.(mg Chl)1.min1in the sink-limited leaves. These results appear to indicatethat the reduction in photosynthetic activity under sink-limitedconditions was mostly due to a deactivation of RuBPcase. Theactivity of deactivated RuBPcase in the sink-limited leaveswas restored to 4.1 µmole CO2.(mg Chl)1.min1by incubation of the enzyme in a medium that contained onlyNa2HPO4. This result suggests that free Pi in chloro-plastsplays an important role in the activation of the enzyme. Thelevel of Pi in the sink-limited leaves was 62% of that in thestandard leaves. On the basis of these various results, it appearsthat the deactivation of RuBPcase in the sink-limited leavesis the result of a decrease in the level of Pi. The role offree Pi in the activation of RuBPcase, in particular at atmosphericconcentrations of CO2, was also investigated. (Received November 30, 1989; Accepted May 11, 1990) 相似文献
14.
Biochemical Limitations to Carbon Assimilation in C3 Plants--A Retrospective Analysis of the A/Ci Curves from 109 Species 总被引:9,自引:0,他引:9
Species-specific differences in the assimilation of atmosphericCO2 depends upon differences in the capacities for the biochemicalreactions that regulate the gas-exchange process. Quantifyingthese differences for more than a few species, however, hasproven difficult. Therefore, to understand better how speciesdiffer in their capacity for CO2 assimilation, a widely usedmodel, capable of partitioning limitations to the activity ofribulose-1,5-bisphosphate carboxylase-oxygenase, to the rateof ribulose 1,5-bisphosphate regeneration via electron transport,and to the rate of triose phosphate utilization was used toanalyse 164 previously published A/Ci, curves for 109 C3 plantspecies. Based on this analysis, the maximum rate of carboxylation,Vcmax, ranged from 6µmol m2 s1 for the coniferousspecies Picea abies to 194µmol m2 s1 forthe agricultural species Beta vulgaris, and averaged 64µmolm2 s1 across all species. The maximum rate ofelectron transport, Jmax, ranged from 17µmol m2s1 again for Picea abies to 372µmol m2 s1for the desert annual Malvastrum rotundifolium, and averaged134µmol m2 s1 across all species. A strongpositive correlation between Vcmax and Jmax indicated that theassimilation of CO2 was regulated in a co-ordinated manner bythese two component processes. Of the A/Ci curves analysed,23 showed either an insensitivity or reversed-sensitivity toincreasing CO2 concentration, indicating that CO2 assimilationwas limited by the utilization of triose phosphates. The rateof triose phosphate utilization ranged from 4·9 µmolm2 s1 for the tropical perennial Tabebuia roseato 20·1 µmol m2 s1 for the weedyannual Xanthium strumarium, and averaged 10·1 µmolm2 s1 across all species. Despite what at first glance would appear to be a wide rangeof estimates for the biochemical capacities that regulate CO2assimilation, separating these species-specific results intothose of broad plant categories revealed that Vcmax and Jmaxwere in general higher for herbaceous annuals than they werefor woody perennials. For annuals, Vcmax and Jmax averaged 75and 154 µmol m2 s1, while for perennialsthese same two parameters averaged only 44 and 97 µmolm2 s1, respectively. Although these differencesbetween groups may be coincidental, such an observation pointsto differences between annuals and perennials in either theavailability or allocation of resources to the gas-exchangeprocess. Key words: A/Ci curve, CO2 assimilation, internal CO2 partial pressure, photosynthesis 相似文献
15.
Differential filtration was used to apportion [32p]orthophosphate(P1) uptake to predominantly bacterial (<3 µm) or algal(>3 µm) components of Lake Kinneret microplankton.Bacteria generally showed preferential 32Pi uptake in comparisonwith algae. Nevertheless, in most cases, the relative proportionof 32P counts retained on 3 µm filters was greater thanthe proportion of 14C counts from heterotrophic bacterial incorporationof [14Clglucose, indicating that algae were competing for Piwith bacteria with some measure of success. Most time courseexperiments did not show any consistent transfer of 32P frombacteria to algae. The addition of a bacterial inhibitor (garamycin)caused a relative increase in the proportion of algal to bacterial32Pi uptake. Added organic P substrates lowered the amount of32Pi uptake and appeared to be preferentially utilized by bacteria.Apparent residence times for Pi in Lake Kinneret ranged from0.4 h (prior to overturn) to 17.4 h during bomothermy. Despitelow ambient Pi concentrations, P limitation in Lake Kinneretis not as extreme as in many other aquatic environments. 相似文献
16.
Yang T Esteve E Pessah IN Molinski TF Allen PD López JR 《American journal of physiology. Cell physiology》2007,292(5):C1591-C1598
Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca2+ microelectrodes in myotubes expressing wild-type RyR1 (WTRyR1) or RyR1 with one of four common MH mutations (MHRyR1), we measured resting intracellular Ca2+ concentration ([Ca2+]i). Changes in resting [Ca2+]i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca2+]i than those expressing WTRyR1. Exposure of myotubes expressing MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca2+]i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca2+]i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca2+]i in myotubes expressing MHRyR1s was significantly greater than that for WTRyR1. From these data, we propose that the high resting myoplasmic [Ca2+]i in MHRyR1 expressing myotubes is due in part to a related structural conformation of MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum 相似文献
17.
Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents 总被引:1,自引:0,他引:1
Secondo A Pannaccione A Cataldi M Sirabella R Formisano L Di Renzo G Annunziato L 《American journal of physiology. Cell physiology》2006,290(1):C233-C243
The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 µM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (IDR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with L-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents 相似文献
18.
Gomora Juan Carlos; Enyeart John J. 《American journal of physiology. Cell physiology》1998,275(6):C1526
Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK+ current(IAC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa2+-dependentcortisol secretion. We studiedIACinhibition byCa2+ and theCa2+ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca2+concentration([Ca2+]i)buffered to 0.02 µM,IAC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca2+]ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca2+]idramatically reducedIAC channelactivity. Ionomycin inhibited IAC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K+ current.Inhibition of IACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.IAC inhibition byionomycin was associated with membrane depolarization. When[Ca2+]iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedIAC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedIAC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca2+]iinhibits IACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withIAC channels. ANGII may inhibitIAC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca2+ asa mediator. 相似文献
19.
Murthy Karnam S.; Makhlouf Gabriel M. 《American journal of physiology. Cell physiology》1998,274(5):C1199
Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa2+-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated45Ca2+release (21 ± 3 to 30 ± 1% decrease in45Ca2+cell content); Ca2+ releasestimulated by 8-BrcGMP was concentration dependent with anEC50 of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa2+ concentration([Ca2+]i)and induced contraction; both responses were abolished after Ca2+ stores were depleted withthapsigargin. With VIP, which normally increases[Ca2+]iby stimulating Ca2+ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca2+]ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca2+ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP3) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa2+ release from anIP3-insensitive store and that itseffect is additive to that of IP3. 相似文献
20.
Ohta Yoshinori; Yoshioka Tatsuya; Mochimaru Mari; Hisabori Toru; Sakurai Hidehiro 《Plant & cell physiology》1993,34(4):523-529
Tentoxin strongly inhibited the ATPase activity of isolatedcoupling factor 1 (AF1) from the cyanobacterium Anacystis nidulans,with 50% inhibition occurring at 0.3 µM. When thylakoidsfrom A. nidulans were preincubated with 0.3 µM tentoxinfor 30 min, photophosphorylation was inhibited by 50%. Measurementsof fluorescence from 9-aminoacridine indicated that tentoxininhibited the utilization of the proton gradient by ATP formationin thylakoids. These results indicate that tentoxin is a strongenergy-transfer inhibitor of photophosphorylation in A. nidulans.Tentoxin decreased the level of ATP in intact cells both inthe light and in darkness, its effects being much stronger inthe dark. Tentoxin at 50 µM strongly inhibited the growthof the cells.
3Present address: Corporate Research and Development Laboratory,Tonen Co. 1-3-1 Nishi-tsurugaoka, Ohi-machi, Saitama, 354 Japan
4Present address: Technology and Engineering Laboratories, AjinomotoCo., Inc. Suzuki-cho 1, Kawasaki, 210 Japan 相似文献