共查询到20条相似文献,搜索用时 15 毫秒
1.
The absence of the thyroid stimulation of hepatic [Na+ + K+]ATPase in obese (ob/ob) mice has been investigated. A wide range of tri-iodothyronine (T3) concentrations failed to enhance the hepatic [Na+ + K+]ATPase of ob/ob mice made hypothyroid by methimazole treatment but glycerophosphate dehydrogenase activity was maximally stimulated at low doses of T3. Adrenalectomy increased the basal activity of [Na+ + K+]ATPase to levels found in lean control mice and restored the response of this enzyme to T3. Body weight gain was unaffected by the induction of hypothyroidism in either lean or ob/ob mice. It is concluded that the adrenal steroids play an important role in the expression of [Na+ + K+]ATPase activity in the ob/ob mouse. 相似文献
2.
We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts. 相似文献
3.
3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography. 总被引:2,自引:0,他引:2
The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system. 相似文献
4.
J S Charnock L P Simonson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,59(3):223-229
1. The specific activity of brain (Na+ + K+)-ATPase and Mg2+ -ATPase of the ground squirrel (Spermophilus richardsonii) is significantly increased after long-term hibernation. 2. The markedly non-linear thermal dependence of (Na+ + K+)-ATPase is unchanged during hibernation whereas the near linear thermal dependence of Mg2+-ATPase undergoes minor alteration after prolonged hibernation. 3. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is significantly decreased after 100 days of hibernation as is both the rate and amount of [3H]-ouabain binding. 4. These changes may be related to alteration in the phospholipid matrix of the membrane rather than alteration in the protein structure of the enzyme. 相似文献
5.
The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction. 相似文献
6.
J S Charnock L P Simonson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1977,58(4):381-387
1. (Na+ + K+)-ATPase from homeotherms and poikilotherms demonstrate non-linear thermal dependence for ATP hydrolysis. Apparent energies of activation from crab nerve preparations are less than those of brain or kidney preparations from beef, rabbit, sheep or ground squirrel. 2. Crab nerve (Na+ + K+)-ATPase is less sensitive to inhibition by ouabain than that from beef or ground squirrel; lower rates of [3H]-ouabain binding and reduced amount of drug bound at equilibrium are found. 3. K+-activated acyl-phosphatase is similar in all preparations. 4. Fluorescence polarization of 12-AS labelled membranes demonstrate greater mobility of crab nerve lipids compared to beef brain which has a thermal transition at 20-25 degrees C. Crab nerve is linear in this range. 相似文献
7.
Michael J McKenna Steve F Fraser Jia L Li Xiao N Wang Michael F Carey Eleonora A Side Judith Morton Gregory I Snell Keld Kjeldsen Trevor J Williams 《Journal of applied physiology》2003,95(4):1606-1616
Lung transplant recipients (LTx) exhibit marked peripheral limitations to exercise. We investigated whether skeletal muscle Ca2+ and K+ regulation might be abnormal in eight LTx and eight healthy controls. Peak oxygen consumption and arterialized venous plasma [K+] (where brackets denote concentration) were measured during incremental exercise. Vastus lateralis muscle was biopsied at rest and analyzed for sarcoplasmic reticulum Ca2+ release, Ca2+ uptake, and Ca2+-ATPase activity rates; fiber composition; Na+-K+-ATPase (K+-stimulated 3-O-methylfluorescein phosphatase) activity and content ([3H]ouabain binding sites); as well as for [H+] and H+-buffering capacity. Peak oxygen consumption was 47% less in LTx (P < 0.05). LTx had lower Ca2+ release (34%), Ca2+ uptake (31%), and Ca2+-ATPase activity (25%) than controls (P < 0.05), despite their higher type II fiber proportion (LTx, 75.0 +/- 5.8%; controls, 43.5 +/- 2.1%). Muscle [H+] was elevated in LTx (P < 0.01), but buffering capacity was similar to controls. Muscle 3-O-methylfluorescein phosphatase activity was 31% higher in LTx (P < 0.05), but [3H]ouabain binding content did not differ significantly. However, during exercise, the rise in plasma [K+]-to-work ratio was 2.6-fold greater in LTx (P < 0.05), indicating impaired K+ regulation. Thus grossly subnormal muscle calcium regulation, with impaired potassium regulation, may contribute to poor muscular performance in LTx. 相似文献
8.
Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor. 相似文献
9.
Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action. 相似文献
10.
Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step. 相似文献
11.
Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus 总被引:9,自引:0,他引:9
S A Ernst J R Palacios G J Siegel 《The journal of histochemistry and cytochemistry》1986,34(2):189-195
Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites. 相似文献
12.
P Braquet N Senn M Fagoo R Garay J P Robin A Esanu E Chabrier T Godfrain 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1986,302(12):443-448
Lignans are natural products, some of which were recently discovered in animal urines, semen and blood plasma. We investigated the actions of animal lignans obtained by total synthesis or extracted from urines of pregnant women on Na+, K+-ATPase in human red cells and human and guinea-pig heart cell membranes. Some of the tested lignans (enterolactone, prestegane B and 3-O-methyl enterolactone) inhibited Na+, K+-pump activity in human red cells with IC50 ranging from 5 to 9 X 10(-4) M. The IC50 for ouabain (7 X 10(-7) M) was not modified by addition of lignans. Enterolactone inhibited Na+, K+-ATPase activity in human and guinea pig heart membranes. It also displaced [3H]-ouabain binding from human heart with IC50 = 1.5 X 10(-4) M. The apparent dissociation rate constants (kd) of [3H]-ouabain were not different in presence of digoxin or enterolactone. Enterolactone exhibited a poor cross reactivity against antidigoxin antibodies. The aglycones of the lignans studied here were slight inhibitors of the Na+, K+-ATPase. However, we cannot exclude that a glycosyl- (and/or butenolide-) derivative of enterolactone could be one "endogenous ouabain-like" factor. 相似文献
13.
ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples. 相似文献
14.
Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.
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Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t). 相似文献
15.
We report on the interactions of Li+, a congener of K+ with the (Na+ + K+)-ATPase from E Electricus as measured by their effects on the rate of [3H]-ouabain binding to this enzyme. Like K+, Li+ slows ouabain binding under both Type I (Na+ + ATP) and Type II (P1) conditions, but with lower affinity. In contrast to K+, the Li+ inhibition curve is hyperbolic, suggesting interaction at an uncoupled site. Also differing from the complete inhibition by high K+, a residual ouabain-binding rate persists at high Li+. The interactions of Li+ and K+ are synergistic: the apparent K+ affinity increases 3 to 4-fold in presence of Li+. These results are consistent with the conclusion that Li+ interacts with only one of the two K+ sites and may be of interest in interpreting lithium pharmacology. 相似文献
16.
J S Charnock L P Simonson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,60(4):433-439
1. The specific activity of renal cortical (Na+ + K+)-ATPase of the Richardson ground squirrel is markedly reduced during hibernation, in contrast to the specific activity of the accompanying Mg2+-ATPase which is markedly increased. 2. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is unchanged by hibernation. 3. Both the non-linear thermal dependence of (Na+ + K+)-ATPase and the linear thermal dependence of Mg2+-ATPase are also unchanged by hibernation. 4. The energy of activation of both enzymes is unchanged during hibernation, or by comparison with that determined in awake controls. 5. There is no evidence for inherent "cold resistance" in these enzyme preparations compared to similar preparations from the non-hibernating rabbit. This parameter does not change during hibernation. 6. Both the rate and amount of specific [3H]-ouabain binding to the renal cortical preparations of (Na+ + K+)-ATPase decrease during hibernation. This decrease matches the fall in enzyme activity so that the ratio of pumping sites/unit of enzyme activity shows no seasonal variations. 7. These findings suggest that the amount of renal cortical (Na+ + K+)-ATPase enzyme falls during hibernation, but that the enzyme which remains functions with the same thermodynamic efficiency and identical biochemical characteristics of that found in the awake summer controls. 相似文献
17.
Further biochemical characterization of an Na+ pump inhibitor purified from human urine 总被引:2,自引:0,他引:2
M Crabos M L Grichois P Guicheney I W Wainer J F Cloix 《European journal of biochemistry》1987,162(1):129-135
An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones. 相似文献
18.
Raiteri L Zappettini S Milanese M Fedele E Raiteri M Bonanno G 《Journal of neurochemistry》2007,103(3):952-961
Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]D-aspartate ([3H] D-ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] 15 mmol/L were prevented by the glutamate transporter inhibitors DL-threo-beta-benzyloxyaspartate (DL-TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+-independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+-evoked release of endogenous glutamate or [3H]D-ASP was inhibited by DL-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked by DL-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia. 相似文献
19.
The cytoplasmic concentrations of Cl-([Cl-]i) and Ca2+ ([Ca2+]i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic beta-cells isolated from ob/ob mice. Steady-state [Cl-]i in unstimulated beta-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl- into beta-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl-]i either in the absence or presence of furosemide inhibition of Na+, K+, 2 Cl- co-transport. Glucose-induced oscillations of [Ca2+]i were transformed into sustained elevation in the presence of 4,4' diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). A similar effect was noted when replacing 25% of extracellular Cl- with the more easily permeating anions SCN-, I-, NO3- or Br-. It is concluded that glucose stimulation of the beta-cells is coupled to an increase in their Cl- permeability and that the oscillatory Ca2+ signalling is critically dependent on transmembrane Cl- fluxes. 相似文献
20.
Thomas Andersen Schmidt Jim Stenfatt Larsen Keld Kjeldsen 《Journal of neurochemistry》1992,59(6):2094-2104
Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles. 相似文献