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Biomedical subjects

E Neher

Publications and source records attributed to E Neher.

At least 109 records · Page 6Linked to original sources
PubMed1989

Regulation of the mannose 6-phosphate/IGF II receptor expression at the cell surface by mannose 6-phosphate, insulin like growth factors and epidermal growth factor.

Mannose 6-phosphate, insulin like growth factors I and II (IGF I, IGF II), insulin and epidermal growth factor (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin fibroblasts. The increase is completed within 10-15 min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the growth factors result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of fibroblasts with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the growth factors, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1989

The patch-clamp technique in the study of secretion.

One of the basic cellular functions of virtually every cell type is the exocytotic release of molecules synthesized, stored and packaged into intracellular vesicles or granules. Over decades much effort has been concentrated on elucidating the chain of events leading to exocytosis. Unfortunately, the nature of the process that ultimately induces membrane fusion is not known, nor has it been established definitively whether or not the final steps in the secretory cascade are identical in different cells. Although the fusion between vesicle and plasma membrane has been neatly documented by electron micrographs, it was only recently that the technique of time-resolved membrane capacitance measurement has provided a more detailed insight into mechanistic aspects of exocytosis, both in terms of the fusion event and the steps involved in stimulus-secretion coupling.

Adrenal Glands↗
PubMed1989

Second messenger-activated calcium influx in rat peritoneal mast cells.

1. To study the regulation of calcium influx in non-excitable cells, membrane currents of rat peritoneal mast cells were recorded using the whole-cell patch-clamp technique. At the same time, intracellular calcium concentration ([Ca2+]i) was monitored via the fluorescent calcium-indicator dye Fura-2, which was loaded into cells by diffusion from the patch pipette. 2. Stimulation of mast cells with secretagogues, such as compound 48/80 or substance P, caused release of Ca2+ from internal stores. In addition, external agonists also induced influx of external calcium in 26% of the cells investigated. The agonist-stimulated Ca2+ influx was increased during membrane hyperpolarization and was associated with small whole-cell currents. 3. Likewise, internal application of inositol 1,4,5-trisphosphate (Ins1,4,5P3:0.5-10 microM) elevated [Ca2+]i due both to release of Ca2+ from internal stores and to influx of external calcium. The Ins1,4,5P3-induced influx was greater at more negative membrane potentials, suggesting that Ins1,4,5P3 opened a pathway through which calcium could enter at a rate governed by its electrochemical driving force. 4. Inositol 1,3,4,5-tetrakisphosphate (Ins1,3,4,5P4) did not induce Ca2+ influx by itself nor did it facilitate or enhance Ins1,4,5P3-induced Ca2+ entry. Calcium influx was also induced by inositol 2,4,5-trisphosphate. Since this inositol phosphate is a poor substrate for Ins1,4,5P3 3-kinase it seems unlikely that Ins1,3,4,5P4 plays a role in the regulation of the Ca2(+)-influx pathway in mast cells. 5. The Ins1,4,5P3-induced Ca2+ influx was associated with whole-cell currents of 1-2 pA or less, with no channel activity detectable in whole-cell recordings. The small size of the whole-cell current suggests either that the Ins1,4,5P3-dependent influx occurs via small-conductance channels that are highly calcium specific or that the influx is not via ion channels. 6. Agonist stimulation also activated large-conductance (ca 50 pS) cation channels, through which divalent cations could permeate; thus, these channels represent a second pathway for Ca2+ influx. The slow speed of activation of the channels by agonists, their activation by internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and the inhibition of agonist activation by internal guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) all suggest that the 50 pS channels are regulated by a second messenger and/or a GTP-binding protein. The activity of the 50 pS channel in mast cells is not sensitive to either Ins1,4,5P3 or Ins1,3,4,5P4. Activity of the channel was inhibited by elevated [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells.

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).

Animals↗
PubMed1989

[The importance of calcium for secretion in excitable and non-excitable cells].

Secretion via exocytosis is a process common to excitable as well as non-excitable cells. The notion that this process is entirely determined by a rise in [Ca]i is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced levels of [Ca]i. It appears appropriate to distinguish between electrically excitable and electrically non-excitable cells. In the former, a rise in [Ca]i is the triggering event for secretion, whereas in the latter, second messengers seem to induce secretion while [Ca]i acts as a modulator of the rate of secretion. Conversely, second messengers may modulate Ca-induced secretion in excitable cells.

Animals↗
PubMed1989

Selective effector coupling of muscarinic acetylcholine receptor subtypes.

Attempts have been made by means of recombinant DNA technology to understand the molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor (mAChR). Molecularly defined mAChR subtypes have been produced from the cloned DNAs in Xenopus oocytes and NG108-15 neuroblastoma-glioma hybrid cells as transient and stable expression systems, respectively, and agonist-induced cellular responses have been examined. The results obtained provide evidence that mAChR subtypes are selectively coupled with different effector systems, albeit not exclusively.

Animals↗
PubMed1988

Intracellular calcium release mediated by two muscarinic receptor subtypes.

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K+-specific conductance of 'small' single-channel amplitude similar to the SK type. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.

Acetylcholine↗
PubMed1988

Regulation of calcium influx by second messengers in rat mast cells.

Biphasic increases in the free intracellular calcium concentration, consisting of a large initial transient followed by a sustained elevation, are frequently observed in non-excitable cells following stimulation. In rat peritoneal mast cells a cAMP- and Ca-activated chloride current can interact with IP3-dependent calcium influx to provide the sustained elevation of intracellular Ca concentration following transient IP3-induced release of calcium from intracellular stores. This novel combination of second messenger systems provides a flexible means to modulate calcium-dependent processes such as exocytosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels.

Lambert-Eaton syndrome, an autoimmune disorder frequently associated with small-cell carcinoma of the lung, is characterized by impaired evoked release of acetylcholine from the motor nerve terminal. Immunoglobulin G (IgG) antibodies from patients with the syndrome, applied to bovine adrenal chromaffin cells, reduced the voltage-dependent calcium channel currents by about 40 percent. When calcium was administered directly into the cytoplasm, however, the IgG-treated cells exhibited normal exocytotic secretion, as assayed by membrane capacitance measurement. Measurement with the fluorescent calcium indicator fura-2 indicated that the IgG treatment reduced potassium-stimulated increase in free intracellular calcium concentration. The pathogenic IgG modified neither kinetics of calcium channel activation nor elementary channel activity, suggesting that a reduction in the number of functional calcium channels underlies the IgG-induced effect. Therefore, Lambert-Eaton syndrome IgG reacts with voltage-dependent calcium channels and blocks their function, a phenomenon that can account for the presynaptic impairment characteristic of this disorder.

Adrenal Glands↗
PubMed1988

Secretory responses of rat peritoneal mast cells to high intracellular calcium.

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.

Animals↗
PubMed1988

Patch-clamp techniques for time-resolved capacitance measurements in single cells.

Two methods are described for estimation of passive cell parameters such as membrane capacitance, membrane conductance and access resistance in tight-seal whole cell recording. Both methods are restricted in their application to cases where the cell under study can be approximated by a simple three-component network with linear properties over some voltage range. One method, referred to as the time domain technique, requires only standard electrophysiological equipment and a computer. Parameters are derived from an analysis of capacitive transients during square wave stimulation. It is readily adaptable to wide variations in experimental parameters. Particularly, it is equally applicable to the "slow whole-cell" configuration (access resistance in the range 100 M omega to 1 G omega) and to normal whole-cell measurements (access resistance typically 10 M omega). The other method applies a sine wave command signal to the cell and employs a lock-in amplifier to analyse the resulting current signal. Two modes of operating the lock-in amplifier are described. One mode provides an output signal directly proportional to small changes in capacitance at maximum resolution (1-10 fF). The other mode, in conjunction with a digital computer, supplies estimates of all passive cell parameters, as does the time domain technique, but with a large amount of data reduction performed by the lock-in amplifier itself. Due to the special hardware, however, this method is not as flexible as the time domain technique.

Animals↗
PubMed1988

Rates of diffusional exchange between small cells and a measuring patch pipette.

(1) Fluorescent compounds and specific ion currents (Na+, K+) were used to study the kinetics of the diffusional exchange between small cells and patch pipettes in the tight seal whole cell configuration of the patch clamp technique. (2) Changes in the intracellular concentration of the test substances following patch rupture could be fitted with single exponentials, provided the access resistance RA of the pipette remained constant during diffusional equilibration. The diffusion time constants were linearly related to the access resistance. (3) When apparent diffusion rates were normalized with respect to access resistance they were found to be dependent on the cell size. However, the cell capacitance, which is proportional to the membrane area turned out not to be a precise measure of the cell size. (4) The experimental diffusion rates changed systematically with the aqueous diffusion coefficient and the inverse third root of the molecular weight. Linear interpolation with respect to these quantities provided estimates of diffusion time constants for the diffusion between patch pipettes and the cytoplasm for substances of interest.

Cell Physiological Phenomena↗
PubMed1988

The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum.

1. Mast cells, isolated from rat peritoneum, were studied under tight-seal, whole-cell recording conditions. Membrane conductance, membrane capacitance and the concentration of free intracellular Ca2+, [Ca2+]i, were measured simultaneously. 2. [Ca2+]i could be accurately buffered to values between 0 and 1.5 microM only if relatively high concentrations of calcium buffers (in the millimolar range) were added to the pipette filling solution against which the cytoplasm was dialysed. At lower buffer concentrations [Ca2+]i was markedly increased by hyperpolarizing the membrane. 3. When added to the pipette, guanosine-3-thio-triphosphate (GTP-gamma-S), a nonhydrolysable analogue of guanosine triphosphate, stimulated a 3.3-fold increase in membrane capacitance, which is indicative of mast cell degranulation (Fernandez, Neher & Gomperts, 1984). 4. In weakly buffered cells, GTP-gamma-S also induced a transient increase in [Ca2+]i which, usually, preceded degranulation. Calcium buffers at 1-5 mM concentration suppressed this transient. 5. High [Ca2+]i alone did not induce degranulation. However, it markedly accelerated GTP-gamma-S-induced degranulation. When [Ca2+]i was buffered to zero, an appreciable fraction of cells degranulated in response to GTP-gamma-S, but very slowly, and only after a long lag phase. 6. Transient increases in [Ca2+]i, evoked either by GTP-gamma-S, or by voltage changes, did not elicit capacitance changes during the lag phase, but accelerated the GTP-gamma-S-induced degranulation response at later times. 7. Internally applied inositol 1,4,5-trisphosphate (IP3) also induced transient increases in [Ca2+]i which did not lead to secretion in the absence of GTP-gamma-S. 8. It is concluded that an increase in [Ca2+]i is neither necessary nor sufficient for secretion from dialysed mast cells. [Ca2+]i, however, acts synergistically with other stimuli to promote secretion. It is the more efficient the more time the other stimulus had been allowed for priming the cell.

Action Potentials↗
PubMed1988

The role of calcium in stimulus-secretion coupling in excitable and non-excitable cells.

Secretion of vesicular contents by exocytosis is a common feature of excitable (neurones, chromaffin cells, beta cells) and non-excitable cells (platelets, neutrophils, mast cells). The simplistic view that the universal mechanism controlling secretion is elevation of [Ca2+]i--whatever the source of this second messenger may be--is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced [Ca2+]i. It is nevertheless clear that in excitable cells an increase in [Ca2+]i is the triggering event that induces secretion. In non-excitable cells, secretion is presumably triggered by other second messengers, although [Ca2+]i appears to act as an important modulator of the rate of secretion. Conversely, these second messenger systems may serve a regulatory function in excitable cells. Given the relative importance of [Ca2+]i in the regulation of cellular functions in excitable and non-excitable cells, it is not surprising that several mechanisms are expressed in these cells to regulate intracellular calcium concentration. The major pathway for Ca2+ in excitable cells is by voltage-activated Ca2+ channels, but release of Ca2+ from intracellular stores, via second messengers, predominates in non-excitable cells, and may also be important in excitable cells. In addition, receptor-operated channels and second messenger-gated conductances may prove to be important. All of these pathways are subject to regulation by a variety of interactive second messenger systems, which provide necessary tuning for an appropriate control of intracellular calcium level.

Animals↗
PubMed1987

Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling.

Transient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to "washout" of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min. The fast "washout" phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed.

Animals↗
PubMed1987

Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels.

Serotonin activates chloride currents in Xenopus oocytes injected with a subfraction of rat brain poly(A)+ mRNA. Patch-clamp recordings from cell-attached patches showed that serotonin, applied locally outside the patch, caused the opening of channels of approximately equal to 3 pS conductance and an average lifetime of approximately equal to 100 msec. The extrapolated reversal potential indicated that the channels are chloride-selective. Single-channel currents with similar characteristics were observed in inside-out patches from native oocytes in response to elevated calcium concentrations on the cytoplasmic side. Measurements of intracellular calcium concentration ([Ca2+]i) by fura-2 fluorescence showed approximately equal to 10-fold increases in [Ca2+]i in response to serotonin application in both normal and calcium-free Ringer solution in mRNA-injected oocytes. Little or no response to serotonin was observed in native oocytes. These results suggest that serotonin activation of receptors that are inserted into the oocyte membrane following injection of rat brain poly(A)+ mRNA can induce calcium release from intracellular stores. The increase in [Ca2+]i subsequently activates calcium-dependent chloride channels. Because calcium-dependent chloride channels and a receptor-controlled mechanism of internal calcium release have been shown to exist in native oocytes, we conclude that the newly inserted serotonin receptors utilized the endogenous second-messenger-mediated calcium release to activate endogenous calcium-dependent chloride channels.

Action Potentials↗