共查询到20条相似文献,搜索用时 15 毫秒
1.
Calvin C. Hale Steven B. Kleiboeker Carol G. Carlton Michael J. Rovetto Chan Jung H. D. Kim 《The Journal of membrane biology》1988,106(3):211-218
Summary Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na–Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na–Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (M
r) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 11 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na–Ca exchange activity. Na–Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na–Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na–Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumM
r of 224,000 Da. Specific45Ca2+-binding SL proteins withM
r of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na–Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na–Ca exchange protein(s) remains incomplete. 相似文献
2.
Ramesh Vemuri Margaret E. Haberland Dora Fong Kenneth D. Philipson 《The Journal of membrane biology》1990,118(3):279-283
Summary We have previously partially purified the sarcolemmal Na+–Ca2+ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K. D., Longoni, S., Ward, R. 1988.Biochim. Biophys.Acta
945:298–306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50–75% of solubilized Na+–Ca2+ exchange activity. The MAb's appear to be reactive with native conformation-dependent expitopes on the Na+–Ca2+ exchanger since they do not react on immunoblots. An indirect method was used to identify Na+–Ca2+ exchange proteins. A column containing Na+–Ca2+ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteinsof, apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our prevous association of Na+–Ca2+ exchange with these proteins. 相似文献
3.
Cherry Ballard Mahmood Mozaffari Stephen Schaffer 《Molecular and cellular biochemistry》1994,135(1):113-119
The signal transduction pathway for insulin-mediated activation of sarcolemmal Na+–Ca2+ exchange was examined. Insulin stimulated Na+–Ca2+ exchanger activity in a dose-dependent manner, with the EC50 being about 0.7 U/l. The insulin effect was blocked by the protein kinase inhibitor, staurosporine, indicating possible involvement of a protein kinase in insulin action. Also, the relationship between the insulin effect and activation of a G protein, was examined by testing the effects of 5 guanylyl imidodiphosphate (Gpp(NH))p) on Na+–Ca2+ exchange in, the presence and absence of insulin. When exchanger activity was assayed at a calcium concentration of 40 M, insulin alone had no effect whereas ATP and Gpp(NH)p increased exchanger activity. However, insulin responsiveness was restored in vesicles preloaded with either ATP or Gpp(NH)p, suggesting that insulin may act through a combination of G protein coupling and protein phosphorylation to enhance Na+–Ca2+ exchanger activity. We conclude that calcium overload in the diabetic heart may involve a defect in acute activation of the exchanger by insulin. 相似文献
4.
Gary Hoel Dr. Mary L. Michaelis William J. Freed J. E. Kleinman 《Neurochemical research》1990,15(9):881-887
Procedures were developed for measurement of Na+/Ca2+ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca2+ in the presence of a Na+ gradient. The kinetic characteristics of the Na+/Ca2+ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The Kact for Ca2+ was estimated to be 32 M for hippocampal and 17 M for cortical tissue. The maximal rate of Ca2+ uptake (Vmax) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na+ gradient, and was optimal in the high pH range. Therefore, membranes in which Na+-dependent o Ca2+ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system. 相似文献
5.
John C. Docherty Thane G. Maddaford Deborah F. Dubo Nicole L. Choptain Grant N. Pierce 《Molecular and cellular biochemistry》1995,144(1):61-66
Tension generation and Ca2+ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na+–Ca2+ exchanger and the Ca2+ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The Km and Vmax of Na+-dependent Ca2+ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [3H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher Kd and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [3H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca2+ channel characteristics in isolated sarcolemmal membranes. 相似文献
6.
Kazuhiro Kikuchi Toshiko Kikuchi Fayez K. Ghishan 《The Journal of membrane biology》1990,114(3):257-265
Summary The developmental maturation of Na+–H+ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na+ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na+ uptake which increased with advancing age. Na+ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na+ uptake was investigated using [amiloride] 10–2–10–5
m. At 10–3
m amiloride concentration, Na+ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK
m for amiloride-sensitive Na+ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV
max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na+–H+ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters. 相似文献
7.
《Biochemical and biophysical research communications》2013,431(2):239-245
Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca2+ current through both enhancement of Ca2+ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na+–H+ exchange (NHE), we examined the possible role of NHE in the regulation of ICa during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm2) fluid was applied onto single rat ventricular myocytes, and the ICa was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the ICa (IC50 = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the ICa in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the ICa under FP was not altered by intracellular high Ca2+ buffering. Replacement of external Cl− with aspartate to inhibit the Cl−-dependent acid loader eliminated the inhibitory effect of HOE642 on ICa. These results suggest that NHE may attenuate FP-induced ICa suppression by preventing intracellular H+ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca2+-dependent inhibition of the ICa during FP exposure. 相似文献
8.
The effect of external and internal K+ on Nao+-dependent Ca2+ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K+ (up to 70 mM) has no effect on Na+?Ca2+ exchange. Removal of Ki+ causes a marked inhibition in the Nao+-dependent Ca2+ efflux component. Internal K+ activates the Na+?Ca2+ exchange with low affinity . Activation by Ki+ is similar in the presence or in the absence of Nai+, thus ruling out a displacement of Nai+ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca2+ efflux on Ki+. The present results demonstrate that Ki+ is an important cofactor (partially required) for the proper functioning of the forward Na+?Ca2+ exchange. 相似文献
9.
Na+−K+ pump activity and the glucose-stimulated Ca2+-sensitive K+ permeability in the pancreatic B-cell 总被引:1,自引:0,他引:1
A rise in the extracellular concentration of glucose from an intermediate to a high value changes the burst pattern of electrical activity of the pancreatic B-cell into a continuous firing, and yet activates the B-cell Ca2+-sensitive K+ permeability. The hypothesis that glucose exerts such effects by inhibiting the Na+, K+-ATPase was investigated. Ouabain (1 mM) mimicked the effect of 16.7 mM glucose in stimulating 86Rb, 45Ca outflow and insulin release from perifused rat pancreatic islets first exposed to 8.3 mM glucose. The stimulation by ouabain of 86Rb outflow was reduced in the absence of extracellular Ca2+ and almost completely abolished in the presence of quinine, and inhibitor of the Ca2+-sensitive K+ permeability. In the presence of ouabain, a rise in the glucose concentration from 8.3 to 16.7 mM failed to stimulate 86Rb outflow. However, the rise in the glucose concentration failed to inhibit 86Rb influx in islet cells, while ouabain dramatically reduced 86Rb influx whether in the presence of 8.3 or 16.7 mM glucose. These findings do not suggest that inhibition of the B-cell Na+, K+-ATPase represents the mechanism by which glucose in high concentration stimulates 86Rb outflow and induces continuous electrical activity in the B-cell. 相似文献
10.
In intact mitochondria supplemented with succinate or -hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na+>Rb+, Cs+, K+, Li+.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K+>Rb+>Cs+>Li+>Na+.When the media are supplemented with phosphate, the Na+/K+ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K+>Na+ with succinate and Na+>K+ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with -hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K+ than in Na+.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH4Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na+/H+ antiporter existent in the membrane. 相似文献
11.
The long arm of chromosome 4D of wheat (Triticum aestivum L.) contains a gene (or genes) which influences the ability of wheat plants to discriminate between Na+ and K+. This discrimination most obviously affects transport from the roots to the shoots, in which less Na+ and more K+ accumulate in those plants which contain the long arm of chromosome 4D. Concentrations of Na+ and K+ in the roots, and Cl− concentrations in the roots and shoots, are not significantly affected by this trait, but Na+, K+ and Cl− contents of the grain are reduced. The trait operates over a wide range of salinities and appears to be constitutive. At
the moment it is not possible to determine accurately the effect of this trait on growth or grain yield because the aneuploid
lines which are available are much less vigorous and less fertile than their euploid parents. 相似文献
12.
Donald M. Bers Kenneth D. Philipson Arthur Peskoff 《The Journal of membrane biology》1985,85(3):251-261
Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m
–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca]. 相似文献
13.
Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease. 相似文献
14.
Richard D. Levere Bruno Escalante Michal Laniado Schwartzman Nader G. Abraham 《Neurochemical research》1989,14(9):861-864
Hemoglobin has been shown to inhibit brain Na+–K+-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na+–K+-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na+–K+-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na+–K+-ATPase. However, in the presence of heme oxygenase, heme inhibited Na+–K+-ATPase by 75%. Pretreatment of rats with SnCl2, a known inducer of heme oxygenase, reduced the basal activity of the brain Na+–K+-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na+–K+-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage. 相似文献
15.
Summary Both simultaneous and consecutive mechanisms for Na+–Ca++ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca++ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na+ ions and one Ca++ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na+Ca++ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m2) and an upper limit for the rate constants of both translocational steps of 105·sec–1, satisfactory behavior was obtainable with identical binding constants for Ca++ on the two sides of the membrane (106
m
–1), similar symmetry also being assumed for the Na+ binding constant (12 to 60m
–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings. 相似文献
16.
James L. Kinsella Timothy Cujdik Bertram Sacktor 《The Journal of membrane biology》1986,91(2):183-191
Summary Na+–H+ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na+], treatment altered the initial rate of Na+ uptake by increasingV
m (hyperthyroid, 18.9±1.1 nmol Na+ · mg–1 · 2 sec–1; normal, 8.9±0.3 nmol Na+ · mg–1 · 2 sec–1), and not the apparent affinityK
Na
+ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H+] and at a subsaturating [Na+] (1mm), hyperthyroidism resulted in the proportional increase in Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H+]0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV
m (hyperthyroid, 1.68±0.14 nmol Na+ · mg–1 · 2 sec–1; normal 0.96±0.10 nmol Na+ · mg–1 · 2 sec–1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H+ translocation or debinding was rate limiting for Na+–H+ exchange since Na+–Na+ exchange activity was greater than Na+–H+ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na+–Na+ and Na+–H+ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na+–Na+ and Na+–H+ exchange activity. 相似文献
17.
《生物化学与生物物理学报:生物膜》1985,817(1):165-173
The orientation of amino groups in the membrane in the α- and β-subunits of (Na+ + K+)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[125I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na+ + K+)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na+ + K+)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of 125I-labeling within the α-subunit. The preservation of (Na+ + K+)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E1- and E2-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface. 相似文献
18.
《生物化学与生物物理学报:生物膜》1987,904(2):353-364
The ATP hydrolysis dependent Na+-Na+ exchange of reconstituted shark (Na+ + K+)-ATPase is electrogenic with a transport stoichiometry as for the Na+-K+ exchange, suggesting that translocation of extracellular Na+ is taking place via the same route as extracellular K+. The preparation thus offers an opportunity to compare the sided action of Na+ and of K+ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K+. With Na+ but no K+ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K+ in concentrations of 50 μM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K+ also gives biphasic ATP-activation curves, however, only when the K+ concentration is 50 mM or higher (Na+ + K+ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E2(Na2+) and E2(K2+), respectively, and in which the dephosphorylation of E2-P is rate limiting in the presence of Na+ (but no K+) extracellular, whereas in the presence of extracellular K+ it is the deocclusion of E2(K2+) which is rate limiting. 相似文献
19.
Glycine (Gly) is considered an obligatory co-agonist at NMDA receptors. Müller glia from the retina harbor functional NMDA receptors, as well as low and high affinity Gly transporters, the later identified as GLYT1. We here studied the regulation of Gly transport in primary cultures of Müller glia, as this process could contribute to the modulation of NMDA receptor activity at glutamatergic synapses in the retina. We demonstrate that neither glutamate stimulation nor the activation or inhibition of protein kinases A or C modify transport. In order to assess a function for Ca2+ and calmodulin (CaM)-dependent processes in the regulation of Gly transport, we explored the participation of Ca2+ concentration, CaM and Ca2+/CaM-dependent enzymes on Gly transporter activity. ATP and carbachol, known to induce Ca2+ waves in Müller cells, as well as caffeine-induced Ca2+ release from intracellular stores stimulated transport, whereas Ca2+ chelation by BAPTA-AM markedly reduced transport. CaM inhibitors W-7, ophiobolin A, R-24571 and trifluoperazine, induced a specific dose-dependent inhibition of transport. The inhibition of CaMKII by the autocamtide-2-related inhibitory peptide or by KN62 caused a decrease in transport which, in the case of KN62, was due to the abolition of the high affinity component, ascribed to GLYT1. Our results further suggest that Gly transport is under cytoskeletal control, as activation of calpain by major increases in [Ca2+]i induced by ionophores, as well as actin destabilization clearly inhibit uptake. We here demonstrate for the first time the participation of CaM, CaMKII and the actin cytoskeleton in the regulation of Gly transport in glia. Ca2+ waves are induced in Müller cells by distinct neuroactive compounds released by neurons and glia, hence the regulation of [Gly] by this system may be of physiological relevance in the control of retinal excitability. 相似文献
20.
Cl– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na+and K+-free internal and external solutions, the basal whole-cell current was predominantly a Cl– current. Cl–-sensitive current (I
Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl–]
o
when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl–] < 40 mm, and was not saturated with a physiological Cl– gradient. The Cl– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl– current. The cAMP- or forskolin-activated and basal Cl– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P
Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca2+]
i
, the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca2+]
i
.This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance. 相似文献