Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10 s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh burst of quanta was also observed at unclamped junctions. At –100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 M TTX to the Ringer's solution, or total replacement of sodium with quanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca²⁺-free, EGTA solutions, or in Ca²⁺-free solutions containing 2 mM Mn²⁺. Sr²⁺ effectively substituted for Ca²⁺. Addition of 2 mM Co²⁺ or Mn²⁺ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl--methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to –100 mV and disappearing at –60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.     

Effect of nitrendipine on histamine release from human basophil leukocytes

The effect of the new calcium antagonist nitrendipine on in vitro basophil activation was evaluated in 10 subjects. The histamine release induced by calcium ionophore A23187, f-met peptide and anti-IgE was inhibited, in a dose-dependent fashion, by nitrendipine in the concentration range of 1-100 microM. The activity of this calcium antagonist seems complex and related to an interference with calcium at multiple sites. At concentrations higher than 200 microM, nitrendipine causes histamine release from basophil leukocytes. This histamine secretion is likely to be due to a cytotoxic effect, since it is associated with an increase in LDH levels in the cell supernatant.     

Age-related reductions in rat atrial high affinity choline uptake, ACh synthesis, and ACh release. A brief note

We have developed a rat atrial mince preparation that can take up choline, acetylate it, and then release acetylcholine in a depolarization-dependent manner. We demonstrate that aging appears to reduce the functional cholinergic activity in this tissue, which may be important for understanding how senescence alters the regulation of cardiac activity.     

Inhibitory Effect of Glucocorticosteroids on Anti-IgE-Induced Histamine Release from Human Basophilic Leukocytes: Evidence for a Dual Mechanism of Action

Anti-IgE-induced histamine release from human leukocytes is inhibited when the cells before challenge are cultured overnight in the presence of glucocorticoids (GCSs). The present report suggests that the GCSs might exert their effect by at least a dual mechanism of action. Histamine release was induced by a suboptimum concentration of anti-IgE. When the release recorded in the presence of the steroid is plotted against the release recorded in its absence, the data points of several experiments fit a regression line characterized by two parameters: its slope and its intercept with the abscissa. Structure-activity examination with selected GCSs indicates that the orders of potency for affecting these two parameters are not identical. Furthermore, pulse experiments suggest that the cells require different times of contact with the steroid to express inhibition according to the two parameters. The removal of adherent cells or platelets did not markedly affect the degree of leukocyte histamine release or its inhibition by a given GCS, suggesting that the steroid interacts directly with the basophil. Finally, steroid-induced inhibition was not affected by the putative phospholipase A2-inhibitor p-bromophenacylbromide (BPB) or the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA).     

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca²⁺]_i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca²⁺ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca²⁺]_i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 M lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca²⁺]_i by stimulating Ca²⁺ release from the sarcoplasmic reticulum independently of extracellular Ca²⁺ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca²⁺ entry through calcium channels was suppressed.     

The paraventricular nucleus of hypothalamus mediates the pressor response to noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats

The medial prefrontal cortex (MPFC) is a structure that is also involved in cardiovascular modulation. The injection of norepinephrine (NE) into the prelimbic (PL) area of the MPFC of unanesthetized rats evokes a pressor response which is mediated by acute vasopressin release. Vasopressin is synthesized by magnocellular cells of the paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the present study, we endeavored to determine which vasopressin-synthesizing hypothalamic nucleus is involved in the pressor pathway activated after NE injection into the PL area of the MPFC. We report here that lidocaine microinjection into the SON did not change the pressor response evoked by NE injection into the PL. However, the response to NE was blocked by prior injection of lidocaine or CoCl₂ into the PVN, indicating that this area is responsible for the mediation of this pressor response. A neuroanatomic experiment in which the neuronal tracer biotinylated dextran amine (BDA) was microinjected into the MPFC showed a lack of axons or neuronal cell bodies in the PVN, indicating that there are no direct connections between the PL area of the MPFC and the PVN. The results suggest that the PVN is involved in the mediation of the pressor response to NE in the PL area and that this pathway must relay in other brain structures before reaching the PVN.     

Effect of influenza A virus on leukocyte histamine release

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.     

Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones

The afterhyperpolarization (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca²⁺-sensitive K⁺ conductances following Ca²⁺ influx via Ca²⁺ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05±3.76% of those evoked at 90-s stimulus intervals (n=35). Since there was no consistent effect of ryanodine (5 M), ruthenium red, (300 M) or dantrolene Na (35 M) on this frequency dependence, it is unlikely to result from release of Ca²⁺ from intracellular stores. Ca²⁺ currents (I _Ca, studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca²⁺-induced I _Ca inactivation and I _Ca run-down. There was excellent correlation (r=0.964) between the estimated changes in Ca²⁺ influx and the expected activation of the Ca²⁺-sensitive K⁺ current, I _AHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of I _Ca.     

The radioimmunoassay of histamine release from circulating basophils in an in vitro study as support for the in vivo active systemic anaphylaxis test in the guinea pig

An in vitro anaphylaxis test is described which explores the ability of compound re-challenge to induce histamine release from polynuclear basophils in whole blood of guinea pigs that had previously been sensitised. This test is used in drug safety, in support of the in vivo active systemic anaphylaxis test, which is sometimes required by regulatory authorities, when the observed clinical signs are not conclusive. This sensitive and discriminative test is inexpensive and reduces animal utilisation.     

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.