Novel chloride conductance in the membrane of bovine chromaffin cells activated by intracellular GTP gamma S.

1. The effects of introducing the non-hydrolysable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) into perfused bovine chromaffin cells were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence [Ca2+]i measurements. 2.

Guanosine 5'-[beta-thio]triphosphate selectively activates calcium signaling in mast cells.

In rat peritoneal mast cells, the activation of GTP-binding proteins (G proteins) by guanosine 5'-[gamma-thio]triphosphate GTP[gamma S] has been found to induce a transient rise in intracellular calcium as well as degranulation. A G protein that couples to phospholipase C (Gp) is thought to mediate the calcium response, whereas degranulation is mediated by a different G protein, termed Ge. In an attempt to activate mast-cell G proteins more selectively, the GTP analogues guanosine 5'-[alpha-thio]triphosphate (GTP[alpha S]) and guanosine 5'-[beta-thio]triphosphate (GTP[beta S]) (RP and SP diastereomers) were introduced into mast cells by means of patch pipettes.

A GTP analogue induces calcium release but not secretion in rat mast cells.

Two G protein-mediated events, calcium release and secretion, were measured in single rat peritoneal mast cells using the patch-clamp technique. Various phosphorothioate analogues of GTP were introduced into the cells. While GTP gamma S and Rp-GTP alpha S activated both processes, Rp-GTP beta S was found to induce repetitive calcium release in the absence of exocytosis.

Fabrication and use of nanometer-sized electrodes in electrochemistry.

Electrodes with electrochemical dimensions as small as 10 angstroms have been fabricated and used for electrochemical studies. These nanometer-scale electrodes have enabled the measurement of electron-transfer rate constants, k(het), that are two orders of magnitude faster than k(het) values accessible with any other electrochemical method.

Effects of angiotensin II on intracellular calcium and electrical function of mouse renal juxtaglomerular cells.

Utilizing a combination of patchclamp and calcium microspectrofluorimetry we have characterized the effects of angiotensin II (Ang II) on intracellular calcium and on the electrical properties of mouse renal juxtaglomerular cells. We found the existence of voltage activated inward and outward rectifying potassium currents, and the inhibition of the anomalous inward rectifying potassium current by Ang II. Blocking the inward rectifyer was paralleled by membrane depolarization, but we obtained no evidence for calcium entry due to voltage-gated calcium channels in JG cells.

Lack of direct evidence for a functional role of voltage-operated calcium channels in juxtaglomerular cells.

In this study we have examined the role of voltage-gated calcium channels in the regulation of calcium in juxtaglomerular cells. Using a combination of patch-clamp and single-cell calcium measurement we obtained evidence neither for voltage-operated calcium currents nor for changes of the intracellular calcium concentration upon acute depolarizations of the cell membrane. Increases of the extracellular concentration of potassium to 80 mmol/l depolarized the juxtaglomerular cells close to the potassium equilibrium potential, but did not alter the intracellular calcium concentration neither in patch-clamped nor in intact Furaester-loaded cells.

Matching-to-sample by an echolocating dolphin (Tursiops truncatus).

An adult male dolphin was trained to perform a three-alternative delayed matching-to-sample task while wearing eyecups to occlude its vision. Sample and comparison stimuli consisted of a small and a large PVC plastic tube, a water-filled stainless steel sphere, and a solid aluminum cone. Stimuli were presented under water and the dolphin was allowed to identify the stimuli through echolocation.

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA.

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle.

Analysis of the published calorimetric evidence for electrochemical fusion of deuterium in palladium.

Estimates are given of the raw data that are the basis for the claims of excess power production by the electrochemical charging of palladium in deuterium oxide (D(2)O). Calorimetric results are also presented that show no anomalous power production in either 0.1M LiOD/D(2)O or 0.

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-.

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.

Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells.

Simultaneous patch-clamp and fura-2 measurements were used to investigate the electrical properties and receptor-mediated changes of intracellular calcium in renal juxtaglomerular cells. Here we report the presence of voltage-activated inward and outward rectifying potassium currents and the inhibition of the anomalous inward rectifying potassium current by angiotensin II (ANG-II). This action of ANG-II was mimicked by guanosine 5'-[gamma-thio]triphosphate but not by cAMP, cGMP, inositol 1,4,5-trisphosphate, or phorbol ester, suggesting that ANG-II inhibits the potassium channel directly by means of a guanine nucleotide-binding regulatory protein or by means of an unusual type of second messenger.

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.

[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.

Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling.

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.

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.

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.

Comparison of target detection capabilities of the beluga and bottlenose dolphin.

The echolocation detection capabilities of a beluga (Delphinapterus leucas) and an Atlantic bottlenose dolphin (Tursiops truncatus) were directly compared in a target detection experiment. Both animals were trained to detect targets in the presence of masking noise. Targets were stainless-steel, water-filled spheres 7.

Propagation of beluga echolocation signals.

The propagation characteristics of high-frequency echolocation signals (peak energies above 100 kHz) of the beluga (Delphinapterus leucas) were measured while the animal performed a target detection task. The whale was trained to station on a bite plate so that its transmission beam could be measured in the vertical and horizontal planes using hydrophone arrays. The transitional region between the acoustic near- and farfields was also located using an array of hydrophones that extended directly in front of the animal in the horizontal plane.

Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals.

(1) We investigated the effects of single- and double-poisoning with tetanus toxin (TeTx), botulinum neurotoxin type A (BoTx A) and botulinum neurotoxin type B (BoTx B) on spontaneous and nerve-evoked quantal transmitter release at motor endplates of the triangularis sterni preparation of the mouse. (2) Inhibitory effects of TeTx and BoTx B on spontaneous and nerve-evoked transmitter release were very similar, except that the action of BoTx B required 500-fold lower concentrations and was less dependent on temperature. BoTx A caused stronger inhibition of quantal release than TeTx or BoTx B, but was comparatively much easier counteracted by 4-aminopyridine (4-AP).

The actions of presynaptic snake toxins on membrane currents of mouse motor nerve terminals.

1. The m. triangularis sterni of the mouse was used to investigate the actions of dendrotoxin, beta-bungarotoxin, crotoxin, taipoxin, bee venom phospholipase A2, aprotinin and apamin on presynaptic currents which flow inside the perineural sheath of nerve bundles upon nerve stimulation.

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.