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1.
Noradrenalin (8 · 10−6 M) and adrenalin (6 · 10−6 and 6 · 10−7 M) were found to cause marked stimulation of short-circuit current (S.C.C.) in isolated toad bladder, but isoprenalin (8 · 10−7 M) was found to be without effect. The percentage rise in S.C.C. due to noradrenalin was found to be inversely proportional to the initial S.C.C. or total conductance of the bladder. Again in the case of noradrenalin the rise in S.C.C. was almost completely abolished by α-adrenergic blockade but not by β-blockade. This rise in S.C.C. was found not to be significantly different from the rise in net Na+ flux. Bidirectional Cl fluxes were estimated using 82Br as a companion radionuclide to 36Cl. No significant net Cl flux was apparent, either before or after addition of any of the three catecholamines tested. However, in some cases the unidirectional Cl fluxes rose markedly following addition of noradrenalin or of adrenalin and this change was not reflected in a change in total conductance. This anomaly was noted to occur in bladders whose initial conductance was of the order of 0.5 kΩ−1 · cm−2 or greater. The evidence presented suggests that two actions of catecholamines on ion transport in toad bladder are (a) to increase Na+ transport via stimulation of α-adrenergic sites and (b) at the concentrations tested to cause an increase in passive Cl permeability in bladders whose initial conductance is high.  相似文献   

2.
The isolated frog skin, bathed with Li+-Ringer (Na+-free) on the outside and Na+-Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li+ influx. The Na+ efflux was 4% of the s.c.c. 10−5 M ouabain depressed Li+ influx and s.c.c. 1010−5 M amiloride abolished the Li+ s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li+-Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li+-Ringer was 29% lower than controls bathed in Na+-Ringer.When the isolated frog skin is bathed in Na2SO4-Ringer it shows electrical rectification which has been correlated with the active transport of Na+. In skins transporting Li+, rectification characteristics are similar to those of skins transporting Na+. When the inner face of the skin is bathed with Li+-Ringer, rectification, PD and s.c.c. decline in a parallel fashion.It is concluded that: (1) Li+ can be transported when Na+ is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li+ and Na+ transport in a similar manner. (3) Li+ transport, like Na+ transport, is associated with rectification. (4) Active transport of Na+ and Li+ seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li+. (5) The cation being transported is not necessarily activating the (Na+-K+-ATPase.  相似文献   

3.
Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10−8 M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1·10−7 M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10−9 M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were Kd1 = (0.82 ± 0.36)·10−10 M and Kd2 = (3.2±0.60)·10−8 M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10−8 M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10−8 M T3 showed a Kd1 = (0.15 ± 0.10)·10−10 M and Kd2 = (3.5 ± 0.10)·10−8 M. We conclude that T3 i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.  相似文献   

4.
The technique of affinity chromatography with the curarizing neurotoxins of Naja naja venom has been employed to extract nicotinic acetylcholine receptors from the brain tissues of mouse and hog. Both carbochol and hexamethonium were used as linear or step gradients to elute the receptor and its properties were investigated in lipid bilayer membranes. Of particular interest is the observation that discrete quanta of conductance could be observed across an NaCl gradient of 1.0:0.1 M. By switching the voltage-clamp across the bilayer between a positive and negative 80 mV, the separate Na+ and Cl conductances of these quanta could be estimated and the following conductances of the smallest discrete quanta were observed: 3.7 · 10−11 Ω−1 (Na+) and 5.9 · 10−11 Ω−1 (Cl) for mouse brain receptors; 3.8 · 10−11 Ω−1 (Na+) and 4.7 · 10−11 Ω−1 (Cl) for hog brain receptors. Large aggregates of receptors appeared to activate and deactivate as multiples of a basic conductance size, although there is evidence that they may not represent the actual gating of ion channels. A “background noise” that is not within the temporal capability of the recording system is also present at an intensity that seems to parallel the number of activated receptors, and in view of recent electrophysiological evidence that the relaxation lifetime of the open channel state is of a millisecond duration, it may be that this “noise” actually represent the channel gating.  相似文献   

5.
Alterations in the state of the membrane lipids affect human red cell K+ transport. Depletion of membrane cholesterol by 29–34% significantly inhibited both total K+ influx and ouabain-sensitive K+ influx. Addition of the hydrophobic anesthetic, chlorpromazine, in concentration from 2 · 10−5 to 2 · 10−4 M increased both total K+ influx and ouabain-sensitive K+ influx. In each case the effect on both processes was almost identical which indicates a linkage between K+ “pump” and “leak”. Further, these results demonstrate that red cell K+ transport can be modulated by local conditions in the micro-environment of the transport system.  相似文献   

6.
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10−8 M and was half-maximal at 7.9±3.4·10−7M. The increase at 1·10−5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10−9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10−5 M dopamine was 2.3±0.9·10−6M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43±0.1·10−7M and 4.7±1.6·10−7M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10/t-7M noradrenaline, 2·10/t-7 M epinine, 4.1±1.8·10/t-6M fluphenazine, 8.0±1.6·10/t-6M haloperidol, 4.2±1.2·10−6Mcis-flupenthixol, 2.7±0.4·10−5Mtrans-flupenthixol, >1·10−5M apomorphine, sulpiride, naloxone and isoproterenol.  相似文献   

7.
In this study, the hydraulic conductivity (Lp), Me2SO permeability ( Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me2SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me2SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me2SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me2SO were plotted against time. Mean (means ± SEM) Lpvalues in the presence of Me2SO ( Me2SOp) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10−2± 0.60 × 10−2, 5.29 × 10−2± 0.40 × 10−2, and 3.69 × 10−2± 0.30 × 10−2μm/min/atm, respectively. The Me2SOvalues were 3.69 × 10−3± 0.3 × 10−3, 1.07 × 10−3± 0.1 × 10−3, 2.75 × 10−4± 0.15 × 10−4, 7.83 × 10−5± 0.50 × 10−5, 5.24 × 10−5± 0.50 × 10−5, and 3.69 × 10−5± 0.40 × 10−5cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (Ea) for Me2SOp, Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.  相似文献   

8.
Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO3, nmol cm− 2 s− 1 − 1,76 ± 0,11control × 1,29 ± 0,08REN 10 μM; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO3, nmol cm− 2 s− 1 − 0.80 ± 0.07control × 0.60 ± 0.06REN 1 μM; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na+/H+exchanger and H+-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.  相似文献   

9.
The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I50) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 · 10−6 to 6.32 · 10−6 M. Benomyl, cambendazole and carbendazim had I50 values from 5.83 · 10−5 to 9.01 · 10−5 M. Thiabendazole had an I50 value of 5.49 · 10−4 M. Inhibitor constants (Ki) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had Ki values of 3.20 · 10−5, 1.73 · 10−5 and 1.10 · 10−4 M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.  相似文献   

10.
Relaxation time measurements T1 and T2 of sodium in Halobacterium halobium pellets were carried out at two frequencies. From those measurements, combined with intensity measurements of the sodium in the system, estimation of the properties of the sodium ions in the system was carried out. It is suggested that three types of sodium ions are present in the bacterial pellet. (A) The extracellular sodium with properties of free solution and (B) sodium which is in the pericellular volume between the cell wall and the cell membrane. There is an exchange between type A and type B sodium. The type B sodium has (e2qQ)/h = 3.7 · 107 rad/s, τcB = 5.2 · 10−6 s and τB = 1 · 10−3 s. The sodium of type C is bound inside the cell and undetected. It's concentration inside the cell is assumed to be 1.9 M.  相似文献   

11.
Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10−4 M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10−5 M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10−4 M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10−6 M, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10−5 M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10−6 M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.  相似文献   

12.
The effects of furosemide and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) on steady-state Cl flux were studied in Ehrlich mouse ascites cells. At 10 mM, furosemide inhibited isotopically-determined Cl flux by 86% without changing cell Cl content, indicating that influx and efflux were depressed by the same amount. These results suggest that at least 86% of the steady-state Cl flux may occur as a one for one exchange. Half of the inhibitory effect was not reversed by vigorous washing with albumin-Ringer. A smaller portion of steady-state Cl flux was inhibited by SITS. The maximum effect of SITS was reached near 0.6 mM; at this concentration Cl flux was reduced by 37% without an alteration in cell Cl content. Possible competition of environment Cl and SITS was investigated by replacing environment Cl with acetate or NO3. These anions reduced the efficacy of SITS because they depressed cell Cl turnover themselves, apparently acting on the same exchange process.  相似文献   

13.
A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10−10−1 · 10−7 M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg2+ and was inhibited by 1 · 10−5 M Ca2+. At sufficiently high concentrations, both ATP (3 mM) and Mg2+ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E1 and E2 at 1 · 10−5 M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.  相似文献   

14.
15.
Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15°C, the response occurred in the 1·10−10−10−7 M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1·10−6 M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.  相似文献   

16.
The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 μM and Vmax of 20 μmol·min−1·mg−1, corresponding values with mixed histones were 12 μM and 1.2 μmol·min−1·mg−1. With both protein substrates the apparent Km for ATP was 11 μM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [γ-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.  相似文献   

17.
Fluorescence redistribution after photobleaching (FRAP) was utilized to select a “fast” lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 ± 1.6) × 10−9 cm2/s and (2.7 ± 2.3) × 10−9 cm2/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 ± 1.9 × 10−9 cm2/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 ± 0.19) × 10−10 cm2/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.  相似文献   

18.
Cells were subjected to a range of 45Ca2+ influx loads with A23187. We measured cell 45Ca2+ with time and A23187 dose, and the apparent 45Ca2+ influxes (≡“J(in,app)”) at Ca2+ steady state. We also measured endogeneous exchangeable and total cell Ca2+, which were 50 and 17–220 μM respectively. The effects of A23187 and Ca2+ on cell ATP, swelling, net Cl permeability, and cell morphology were measured. These were modest and do not affect our conclusions.J(in,app) 3 × 10−4 [A23187]2.9·[Ca2+(o)]μmoles/l·min with 92–552 μM [Ca2+(o)] (≡ external Ca2+ concentration) and 0–7 μM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca2+ crossing the membrane is expelled by the pump before it can move deeper into the cell.Calcium pumping increased rapidly in response to increased influx, but the steady state cell 45Ca2+ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 μmoles/l·min with 184 μM [Ca2+(o)]. This is not the result expected from a simple feedback mechanism.At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium 45Ca2+ and [A23187]−2. From the plot we calculated α≡free/total exchangeable Ca2+ = 0.38 ± 0.08 (n = 3) and a maximum pump rate, “Pmax” = 78 μmole/l·min. Pmax is underestimated insofar as J(in,app) is less than the true influx.  相似文献   

19.
The role of prostaglandins (PGs) in the mechanism of action of acetylcholine (ACh) on frog adrenocortical cells has been examined. Administration of a single dose of ACh (5 × 10−5 M) to perifused frog interrenal fragments, for 20 min, stimulated the production of corticosterone, aldosterone, PGE2 and 6-keto-PGF. In contrast ACh did not significantly alter TXB2 production. The effect of ACh could be mimicked by muscarine (10−5 M). Conversely, nicotine (10−6 to 10−4 M) was totally inactive. The increase in PG biosynthesis preceded the peak of corticosteroid release. Repeated 20-min pulses of ACh (5 × 10−5 M) or muscarine (10−5 M) given at 130-min intervales induced a desensitization phenomenon. In presence of indomethacin (5 × 10−6 M), the effect of ACh on PG and steroid secretion was totally abolished. In calcium-free medium, the effect of ACh on PG and corticosteroid production was completely blocked. These results indicated that, in the frog, ACh stimulates corticosteroid secretion through a PG-dependent mechanism.  相似文献   

20.
Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T1 relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10−6 cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10−8 cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10−11cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.  相似文献   

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