Summary The characteristics of the increased calcium (Ca) influx observed in metabolically depleted red blood cells (RBCs) of hypertensive patients were investigated. Twenty-four normotensives, 16 untreated essential hypertensives, and 10 essential hypertensives under sufficient blood pressure control by 50–100 mg/day atenolol were studied. Free intracellular concentrations of Ca, sodium (Na), and potassium (K) were assessed using ion-selective electrodes in freeze-thawed RBCs, which were metabolically depleted by 30 mM desoxy-glucose at 37°C for 48 h. In the treated hypertensives values for Ca and K at 24 and 48 h were not different from values for the normotensives, whereas elevated Ca was found in RBCs of untreated hypertensives. Na in treated hypertensives was significantly increased at 0 and 48 h, thus, being similar to values for untreated hypertensives. Additionally, RBCs of six normals were stressed in a glass/teflon potter. Before metabolic depletion electrolytes were not affected by this procedure, while Ca at 24 and 48 h of metabolic depletion increased to significantly higher values for the hypertensive patients as compared to the controls. These results suggest that the altered Ca metabolism in the RBCs of hypertensives may reflect a secondary phenomenon due to the mechanical damage to RBCs by the elevated blood pressure. 相似文献
The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05–0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca2+ concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflamatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1–1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K+ current and Ca2+-activated K+ current in a concentration-dependent manner. Niflumic acid (0.1–1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na+ current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K+ currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca2+ with each impulse. 相似文献
We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties
of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor
c-Fms, activates a K+ current that is absent in control cells. The activation of the K+ current was Ca2+-dependent, voltage-independent, and was completely blocked by the K+ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC50 values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K+ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding
site for the Ras-GTPase-activating protein led to a complete abolishment of the K+ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the
growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity
of v-Fms, led to a 55% reduction of the K+ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the
K+ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK
can modulate K+ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to
the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our
data suggest that Ras plays a key role for K+ channel activation by the oncogenic RTK v-Fms.
Received: 19 November 1997 / Accepted: 21 January 1998 相似文献
White matter is a compact structure consisting primarily of neuronal axons and glial cells. As in other parts of the nervous system, the function of glial cells in white matter is poorly understood. We have explored the electrophysiological properties of two types of glial cells found predominantly in white matter: type 2 astrocytes and oligodendrocytes. Whole-cells and single-channel patch-clamp techniques were used to study these cell types in postnatal rat optic nerve cultures prepared according to the procedures of Raff et al. (Nature, 303:390-396, 1983b). Type 2 astrocytes in culture exhibit a "neuronal" channel phenotype, expressing at least six distinct ion channel types. With whole-cell recording we observed three inward currents: a voltage-sensitive sodium current qualitatively similar to that found in neurons and both transient and sustained calcium currents. In addition, type 2 astrocytes had two components of outward current: a delayed potassium current which activated at 0 mV and an inactivating calcium-dependent potassium current which activated at -30 mV. Type 2 astrocytes in culture could be induced to fire single regenerative potentials in response to injections of depolarizing current. Single-channel recording demonstrated the presence of an outwardly rectifying chloride channel in both type 2 astrocytes and oligodendrocytes, but this channel could only be observed in excised patches. Oligodendrocytes expressed only one other current: an inwardly rectifying potassium current that is mediated by 30- and 120-pS channels. Because these channels preferentially conduct potassium from outside to inside the cell, and because they are open at the resting potential of the cell, they would be appropriate for removing potassium from the extracellular space; thus it is proposed that oligodendrocytes, besides myelinating axons, play an important role in potassium regulation in white matter. The conductances present in oligodendrocytes suggest a "modulated Boyle and Conway mechanism" of potassium accumulation. 相似文献
Summary Whole cell voltage-clamp recordings from GH3 cells, a clonal cell line derived from a rat anterior pituitary tumor, demonstrated a rapidly activating and inactivating (transient) voltage-dependent outward current. This current, referred to as IA, was elicited by step depolarization from holding potentials negative to –50 mV, showed strong outward rectification at potentials positive to –30 mV, and exhibited steady state inactivation with V1/2 near –64 mV. The current rose to a peak within < 10–20 ms following depolarization and decayed in two exponential phases, IAf and IAAS with time constants of 30–50 and 500–700 ms, respectively. Both IA components exhibited similar voltage dependencies for activation and inactivation. Aminopyridines (2 mol/l – –5 mmol/l) produced a dose dependent, reversible blockade of IA (70% inhibition at 0.5 to 2 mmol/l) with the following rank order of potencies: 4-aminopyridine > 3,4-diaminopyridine = 3-aminopyridine > 2-aminopyridine. These drugs reduced the peak conductance of IA, and produced complex effects on its time-dependent decay. With submaximal degrees of block, there was an increase in the inactivation rate, suggesting that open channels are preferentially blocked by the drugs. It is concluded that GH3 pituitary cells possess an aminopyridine-sensitive transient outward current comparable to the A-current in neural cells. However, this cell line is unusual in that it expresses both rapidly and slowly decaying A-current components.Abbreviations
n-AP
n-aminopyridine
- 3,4-DAP
3,4-diaminopyridine
- TEA
tetraethylammonium
- EGTA
ethylene glycol bis(-aminoethyl ether)N,N-tetraacetic acid
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Send offprint requests to M. A. Rogawski at the above address 相似文献
The effect of beta1 and beta2 blockade on dopamine and dobutamine induced hypokalaemia was investigated in dogs. Arterial plasma potassium was continuously monitored using a potassium-sensitive electrode sited in the abdominal aorta. Dopamine and dobutamine were infused at 15g kg-1 min-1 into animals pre-treated with atenolol (0.5 mg kg-1) or ICI 118551 (0.5 mg kg-1) and the potassium changes compared to those seen in control animals. The inotrope infusions caused a small and transient rise in plasma potassium (p<0.05) followed by a more profound hypokalaemia (p<0.05) sustained for the duration of the infusion. The potassium falls in the control animals were similar to those in the beta1 and beta2 blockade groups. The results suggest that beta2 receptor stimulation cannot alone be responsible for dopamine and dobutamine induced hypokalaemia and a multiple receptor activation is postulated.M. Smith and H.F. Drake were supported by a grant from the Sir Jules Thorn Charitable Trust 相似文献
Summary The action of the potassium channel activator, cromakalim (BRL 34915), on membrane potential, input resistance and current-voltage-relationship of CA3 neurons in a slice preparation of the guinea-pig hippocampus was investigated by means of intracellular recordings. In the presence of tetrodotoxin, cromakalim (30–100 mol/l) produced a hyperpolarization up to 4 mV associated with a decrease in input resistance up to 10 MOhms. Determination of the equilibrium potential of the cromakalim action revealed that the hyperpolarization is due to the activation of a potassium conductance. This cromakalim-activated potassium conductance was voltage-dependent, i.e. it increased with hyperpolarization. Among a number of potassium channel blockers tested, only Cs+ (2 mmol/l) and Ba2+ (0.5 mmol/1) were able to inhibit the cromakalim-induced effects. Simultaneously, both cations suppressed the hyperpolarizing inward rectification (anomalous rectification) in these neurons, indicating that cromakalim activated or potentiated an inwardly rectifying potassium conductance. In addition, cromakalim slightly enhanced both amplitude and duration of afterhyperpolarizations following single calcium-dependent action potentials, suggesting that cromakalim might have a weak facilitatory effect on calcium-dependent potassium conductances.Send offprint requests to C. Alzheimer at the above address 相似文献
The inhibitory effect of a high external Ca2+ ([Ca2+]o) on spontaneous transmitter release in a high K+ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca2+ ([Ca2+]i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca2+]i; increasing [Ca2+]o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K+ concentration (7–15 mM), their facilitatory effects on m.e.p.p. frequency decreased, disappeared and eventually reversed to depressant actions. Since a rise in the external K+ concentration would increase the steady state level of [Ca2+]i, it is suggested that when the [Ca2+]i is preset at a high level, manipulations so as to further increase [Ca2+]i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca2+]i. 相似文献
The intracellular antibody technology has many applications for proteomics studies.
The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.
We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.
The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome. 相似文献