PT pretreatment inhibits 48/80-induced activation of Ca(+)-permeable channels in rat peritoneal mast cells

1990 ◽  
Vol 259 (5) ◽  
pp. C715-C722 ◽  
Author(s):  
M. Kuno ◽  
J. Kawaguchi ◽  
M. Mukai ◽  
F. Nakamura
Keyword(s):  
Mast Cells ◽  
Patch Clamp ◽  
Room Temperature ◽  
Plasma Membranes ◽  
Patch Clamp Technique ◽  
Permeable Channel ◽  
Peritoneal Mast Cells ◽  
Fluorescence Change ◽  
Stimulus Secretion Coupling ◽  
Rat Peritoneal Mast Cells

We recently reported that the secretagogue, compound 48/80, activated Ca2(+)-permeable channels of mast cells possibly via a second messenger [Kuno, Okada, and Shibata. Am. J. Physiol. 256 (Cell Physiol. 25): C560-C568, 1989]. The effects of pretreatment with pertussis toxin (PT) on compound 48/80 (48/80)-induced activation of the Ca2(+)-permeable channel have now been investigated by measuring Ca2+ signals of cell suspensions using the Ca2+ indicator fura-2 and by recording Ba2+ currents through the channel using the patch-clamp technique. In the presence of extracellular Ca2+, the fluorescence change was biphasic, with an immediate rise and a delayed peak at room temperature. The delayed peak, mainly due to Ca2+ entry through plasma membranes, was greatly reduced by pretreatment with PT. The quenching of the fluorescence by 48/80-induced Mn2+ influx was also decreased by PT, whereas the Ca2+ transients due to Ca2+ release from the intracellular stores apparently did not change. In patch-clamp recordings from cell-attached patches with pipettes containing isotonic Ba2+, the 48/80-induced Ba2+ currents were either suppressed or delayed in the PT-treated cells, under conditions where degranulation was absent. These results suggest that PT-sensitive GTP-binding protein is involved in activating the Ca2(+)-permeable channel in mast cells during stimulus-secretion coupling.

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

1987 ◽  
Vol 7 (4) ◽  
pp. 313-321 ◽  
Author(s):  
Reinhold Penner ◽  
Michael Pusch ◽  
Erwin Neher
Keyword(s):  
Mast Cells ◽  
Calcium Transients ◽  
Capacitance Measurement ◽  
Patch Clamp Technique ◽  
Peritoneal Mast Cells ◽  
Cell Recording ◽  
Stimulus Secretion Coupling ◽  
Rat Peritoneal Mast Cells ◽  
Indicator Dye ◽  
Signal Mediators

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.

scholarly journals A patch-clamp study of histamine-secreting cells.

1986 ◽  
Vol 88 (3) ◽  
pp. 349-368 ◽  
Author(s):  
M Lindau ◽  
J M Fernandez
Keyword(s):  
Mast Cells ◽  
Patch Clamp ◽  
Single Channel ◽  
Resting Potential ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Peritoneal Mast Cells ◽  
Ionic Conductances ◽  
Rat Peritoneal Mast Cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.

scholarly journals Evidence for a role of phosphatidylinositol turnover in stimulus–secretion coupling. Studies with rat peritoneal mast cells

1979 ◽  
Vol 178 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Shamshad Cockcroft ◽  
Bastien D. Gomperts
Keyword(s):  
Mast Cells ◽  
Concanavalin A ◽  
Immunoglobulin E ◽  
Histamine Secretion ◽  
Phosphatidylinositol Turnover ◽  
Phosphatidylinositol Response ◽  
Peritoneal Mast Cells ◽  
Stimulus Secretion Coupling ◽  
Rat Peritoneal Mast Cells

Histamine secretion and phosphatidylinositol turnover were compared in antigen-sensitized rat peritoneal mast cells stimulated with a number of different ligands. A small and variable increase in the incorporation of [32P]Pi and of [3H]inositol into phosphatidylinositol was observed when the cells were treated with immunoglobulin E-directed ligands (antigens and concanavalin A), and this was accompanied by a low amount of secretion (<10% of total cell histamine). In the presence of added phosphatidylserine, the addition of immunoglobulin E-directed ligands invariably led to an enhanced rate (approx. 4-fold) of labelling of phosphatidylinositol and, in the presence of Ca2+, this was accompanied by the secretion of histamine. The labelling of phosphatidylinositol and histamine secretion were also stimulated by chymotrypsin and compound 48/80. Whereas the phosphatidylinositol response did not require the presence of extracellular Ca2+, the secretion of histamine was either enhanced or dependent on extracellular Ca2+ (depending on the ligand used). The dependence on ligand concentration for the phosphatidylinositol response and histamine secretion were similar. The increased isotopic incorporation into phosphatidylinositol continued for about 1h although histamine secretion (elicited with concanavalin A) stopped within 2min. These results support the proposition that metabolic events involving phosphatidylinositol play a necessary intermediate role in the regulation of Ca2+ channels by ligand-activated receptors.

A patch-clamp study: secretagogue-induced currents in rat peritoneal mast cells

1989 ◽  
Vol 256 (3) ◽  
pp. C560-C568 ◽  
Author(s):  
M. Kuno ◽  
T. Okada ◽  
T. Shibata
Keyword(s):  
Mast Cells ◽  
Patch Clamp ◽  
Reversal Potential ◽  
Whole Cell ◽  
Current Voltage ◽  
Peritoneal Mast Cells ◽  
Induced Currents ◽  
Rat Peritoneal Mast Cells ◽  
Whole Cell Recordings ◽  
Current Voltage Relation

Ca2+ entry through plasma membrane has been considered to play a significant role in elevating cytosolic free Ca2+ concentrations during stimulus-secretion coupling in mast cells, but electrophysiological evidence of the Ca2+ channels is lacking. We examined the properties of secretagogue (compound 48/80)-induced currents in rat peritoneal mast cells, using the patch-clamp technique. In the whole cell recordings, the addition of compound 48/80 induced transient currents that were suppressed by Cd or reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In Ringer solution containing 2 mM Ca2+, the current-voltage relation was fairly linear from -100 to 50 mV and the reversal potential was 14 +/- 10.1 mV (n = 9). When the external Ca2+ was approximately 1 microM, the compound 48/80-induced currents were marginal, but readmission of Ca2+ or Ba2+ to the bath solution led to an appearance of the currents. In the cell-attached patches, the stimulation enhanced the activity of inward current mediated by Ba2+. The unitary inward Ba2+ current was characterized by the unitary conductance of 10.5 +/- 2.0 pS (n = 10) with isotonic BaCl2 pipette solution, the extrapolated reversal potential of 60.7 +/- 16.0 mV (n = 10) positive to the resting membrane potentials. The percent open time of the inward Ba2+ current channel was not appreciably changed by voltage. In some whole cell recordings, an increase in openings of the cation-selective channel (20-45 pS) was identified in the stimulated cells. When the external Na+ was completely replaced by choline+, the compound 48/80-induced currents had a fairly linear current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)

Clarithromycin Dose-Dependently Stabilizes Rat Peritoneal Mast Cells

Chemotherapy ◽  
2016 ◽  
Vol 61 (6) ◽  
pp. 295-303 ◽  
Author(s):  
Itsuro Kazama ◽  
Kazutomo Saito ◽  
Asuka Baba ◽  
Tomohiro Mori ◽  
Nozomu Abe ◽  
...  
Keyword(s):  
Mast Cell ◽  
Plasma Membrane ◽  
Mast Cells ◽  
Water Soluble ◽  
Patch Clamp Technique ◽  
Membrane Deformation ◽  
Whole Cell Patch Clamp ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells ◽  
First Time

Background: Macrolides, such as clarithromycin, have antiallergic properties. Since exocytosis in mast cells is detected electrophysiologically via changes in membrane capacitance (Cm), the absence of such changes due to the drug indicates its mast cell-stabilizing effect. Methods: Employing the whole-cell patch clamp technique in rat peritoneal mast cells, we examined the effects of clarithromycin on Cm during exocytosis. Using a water-soluble fluorescent dye, we also examined its effect on deformation of the plasma membrane. Results: Clarithromycin (10 and 100 μM) significantly inhibited degranulation from mast cells and almost totally suppressed the GTP-γ-S-induced increase in Cm. It washed out the trapping of the dye on the surface of mast cells. Conclusions: This study provides for the first time electrophysiological evidence that clarithromycin dose-dependently inhibits the process of exocytosis. The mast cell-stabilizing action of clarithromycin may be attributable to its counteractive effect on plasma membrane deformation induced by exocytosis.

Exocytosis (Secretory Granule Extrusion) Induced by Injection of Calcium into Mast Cells

1973 ◽  
Vol 51 (12) ◽  
pp. 1001-1004 ◽  
Author(s):  
T. Kanno ◽  
D. E. Cochrane ◽  
W. W. Douglas
Keyword(s):  
Mast Cells ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Intracellular Concentration ◽  
General Requirement ◽  
Peritoneal Mast Cells ◽  
Direct Support ◽  
Ca Ions ◽  
Stimulus Secretion Coupling ◽  
Rat Peritoneal Mast Cells

Injection of Ca (but not Mg or K) through micropipettes placed within rat peritoneal mast cells elicited extrusion of secretory granules. The result is considered direct support for the view that a rise in the intracellular concentration of Ca ions suffices to induce exocytosis and accounts for the general requirement for Ca in stimulus–secretion coupling.

scholarly journals ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 459-476 ◽  
Author(s):  
P E Tatham ◽  
M Lindau
Keyword(s):  
Mast Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Permeabilized Cells ◽  
Cation Selectivity ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells ◽  
Gamma S

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

Inhibitors of the arachidonic acid cascade dissociate 48/80-induced Ca2+ influx and Ca2+ release in mast cells

1993 ◽  
Vol 264 (4) ◽  
pp. C912-C917 ◽  
Author(s):  
M. Kuno ◽  
J. Kawawaki ◽  
T. Shibata ◽  
H. Gotani
Keyword(s):  
Arachidonic Acid ◽  
Mast Cells ◽  
Nordihydroguaiaretic Acid ◽  
Arachidonic Acid Cascade ◽  
Lipoxygenase Inhibitor ◽  
Low Concentrations ◽  
Peritoneal Mast Cells ◽  
Stimulus Secretion Coupling ◽  
Rat Peritoneal Mast Cells ◽  
Full Activation

Effects of inhibitors of the arachidonic acid cascade on Ca2+ release from intracellular stores and Ca2+ influx through the plasma membrane during stimulus-secretion coupling were examined using rat peritoneal mast cells loaded with fura-2. Compound 48/80 (48/80) was used as a secretagogue. A phospholipase inhibitor, p-bromophenacyl bromide (PBPB), or a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), inhibited the 48/80 (1 microgram/ml)-induced release of histamine, Ca2+, and Mn2+ influxes, but the cyclooxygenase inhibitor, indomethacin (approximately 50 microM), inhibited neither Ca2+ nor Mn2+ influxes. The Ca2+ release induced by 1 microgram/ml of 48/80 was little inhibited by PBPB, NDGA, or indomethacin. The Ca2+ release was activated and saturated with lower concentrations of 48/80 than was the Ca2+ influx. The percent inhibition of the Ca2+ release by 25 microM PBPB was increased by lowering the concentration of 48/80, but NDGA (10 microM) did not inhibit the Ca2+ release induced by low concentrations of 48/80 (0.03-0.1 microgram/ml). These results suggest that activation of the Ca2+ release and the Ca2+ influx were differently regulated and that full activation of Ca2+ influx needs the arachidonic acid cascade produced by higher concentrations of 48/80 than does the Ca2+ release. Lipoxygenase metabolites of arachidonic acid are potential modulators of the Ca2+ influx.

scholarly journals α1-Adrenergic Receptor Blockade by Prazosin Synergistically Stabilizes Rat Peritoneal Mast Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Nozomu Abe ◽  
Hiroaki Toyama ◽  
Yutaka Ejima ◽  
Kazutomo Saito ◽  
Tsutomu Tamada ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Receptor Antagonist ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
High Dose ◽  
Patch Clamp Technique ◽  
Receptor Agonists ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells

Background. Adrenaline quickly inhibits the release of histamine from mast cells. Besides β2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. Methods. Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. Results. Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a β2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high-dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells. Conclusions. This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by β2-adrenergic receptors.

Sign in / Sign up

Export Citation Format

Share Document