Role of acid sphingomyelinase in the regulation of mast cell function.

BACKGROUND: Degranulation of mast cells is stimulated by store-operated Ca(2+) -entry (SOCE). In other cell types, Ca(2+) -entry is modified by ceramide. Exogenously added ceramide has been shown to trigger mast cell apoptosis. Effects of endogenously produced ceramide in mast cells remained, however, elusive. Ceramide may be produced from sphingomyelin by acid sphingomyelinase (Asm). OBJECTIVE: This study explored the impact of Asm on mast cell functions. METHODS: Mast cells were isolated from bone marrow (BMMCs) or peritoneal lavage of gene-targeted mice lacking Asm (asm(-/-)) and their wild-type littermates (asm(+/+)). BMMC maturation and apoptosis-associated annexin V binding were determined by flow cytometry. Asm activity was assessed enzymatically, cytosolic Ca(2+) activity ([Ca(2+)]i) utilizing Fura-2 fluorescence, current across the cell membrane by whole-cell patch clamp, degranulation from hexosaminidase-release and migration utilizing a transwell chamber. In vivo anaphylaxis was derived from decrease in body temperature. RESULTS: Peritoneal mast cell number, BMMC phenotype, spontaneous BMMC apoptosis as well as BMMC CD117, CD34 and FcεRI expression were similar in both genotypes. In asm(+/+) BMMCs, stimulation with antigen resulted in a fast ~2.5-fold increase in Asm activity. Release of Ca(2+) from internal stores and hence several Ca(2+) -dependent functions were strongly impaired in asm(-/-) BMMCs. Thus, antigen-induced increase in [Ca(2+)]i in IgE-sensitized cells, antigen- but not ionomycin-induced currents through Ca(2+) -activated K(+) -channels (KCa 3.1), IgE/antigen-triggered ß-hexosaminidase release, and antigen-induced migration were all lower in asm(-/-) BMMCs than in asm(+/+) BMMCs. Pharmacological inhibition of Asm by amitriptyline (500 nm, 3 h) in asm(+/+) BMMCs similarly decreased antigen-induced increase in [Ca(2+)]i , KCa 3.1 currents, ß-hexosaminidase release and migration. The decrease in body temperature upon the induction of systemic anaphylaxis was significantly less pronounced in asm(-/-) mice than in asm(+/+) mice, an observation pointing to in vivo significance of Asm. CONCLUSIONS AND CLINICAL RELEVANCE: Asm is a novel, powerful regulator of mast cell function and thus a potential target in the treatment of allergic reactions.

Electrical properties of a light-addressable microelectrode chip with high electrode density for extracellular stimulation and recording of excitable cells.

A light-addressable microelectrode chip with 3600 TiN electrodes was fabricated. Amorphous silicon (a-Si:H) serves as a photo conductor. The electrodes on the chip are addressed by a laser spot and electrical properties of the system are determined. DC measurements show a dark to bright dynamic of 10(6)-10(7). The AC impedance dynamic @ 1 kHz/100 mV and thus the signal-to-noise-ratio is determined to 60. This value is quite sufficient for electrophysiological measurements. For the first time, recordings from cardiac myocytes are reported using the principle of light-addressing. Measurements were done with a standard laser scan microscope (Zeiss LSM 410).

Molecular basis of dark adaptation in rod photoreceptors.

Following exposure of the eye to an intense light that 'bleaches' a significant fraction of the rhodopsin, one's visual threshold is initially greatly elevated, and takes tens of minutes to recover to normal. The elevation of visual threshold arises from events occurring within the rod photoreceptors, and the underlying molecular basis of these events and of the rod's recovery is now becoming clearer. Results obtained by exposing isolated toad rods to hydroxylamine solution indicate that, following small bleaches, the primary intermediate causing elevation of visual threshold is metarhodopsin II, in its phosphorylated and arrestin-bound form. This product activates transduction with an efficacy about 100 times greater than that of opsin.

Rod pigment and rod noise in the European toad Bufo bufo.

Behavioural experiments and ganglion cell recordings indicate that the visual sensitivity of dark-adapted toads is limited by the occurrence of spontaneous isomerization-like noise events in the rods. The frequency of these "false photons" has previously been studied (with micropipette recording) in the toad species Bufo marinus, while the behavioural thresholds were determined using Bufo bufo toads. Thus, it was necessary to check that the noise event frequency is roughly the same in these two species. Here we show that it is, in both species, close to 0.02 events per second and rod (at 22 degrees C). Using microspectrophotometry we further show that the absorption spectra of these two rhodopsins are very similar, peaking around 503.3 and 501.8 nm for B. marinus and B. bufo, respectively.

Effect of hydroxylamine on photon-like events during dark adaptation in toad rod photoreceptors.

1. The suction pipette technique was used to investigate the recovery of toad rod photoreceptors following small bleaches of 0.2-3% of the rhodopsin. 2. The reduction in sensitivity and the increase in noise elicited by bleaches were measured, and from these measurements the underlying rate of occurrence of photon-like events was calculated as a function of time after the bleach. 3. Exposure to hydroxylamine solution was used to hasten the decomposition of the metarhodopsin photoproducts. The outer segment was exposed to 110 mM hydroxylamine in a low-Ca2+ Ringer solution for a period of 10-50 s beginning 10-17 min after the bleaching exposure. 4. By the time of the hydroxylamine exposure, the flash sensitivity and response kinetics had returned almost to normal, and were not significantly altered by the exposure. 5. Following hydroxylamine exposure, the rate of spontaneous photon-like events in the rods declined rapidly to near pre-bleach levels. 6. We conclude that hydroxylamine reduces the rate of occurrence of photon-like events induced by a bleach, and we postulate that this reduction results from the removal of metarhodopsin (most likely metarhodopsin II) from the outer segment. 7. Our results are consistent with a model in which photon-like events result from reversal of the reactions (phosphorylation and capping by arrestin) that lead to inactivation of the activated form of rhodopsin, Rh*.

Force dependent response reversal mediated by low- and high-threshold afferents from the same mechanoreceptor in a crayfish leg.

1. The reflex responses initiated by cuticular stress detector one (CSD1) afferents in anterior levator motor neurnons in the fifth walking leg of crayfish were studied in an in vitro preparation by means of intracellular recordings. 2. The response in the anterior levator reversed with strong mechanical stimulation. Inhibitory responses were correlated with the activation of low-threshold CSD1 units, excitatory responses with the activation of high-threshold CSD1 units. Two opposing reflexes thus originate from the same sense organ at different levels of stimulation. 3. Some low-threshold units were inactive at stimulus strengths that activated the high-threshold units. Other low-threshold units remained active. Their reflex effects were reduced by either pre- or postsynaptic mechanisms. 4. Functionally, this force dependent response reversal could be important in switching from postural/locomotor responses to defensive responses such as limb autotomy.

Synaptic connections of the cuticular stress detectors in crayfish: mono- and polysynaptic reflexes and the entrainment of fictive locomotion in an in vitro preparation.

The reflex connections made by Cuticular Stress Detector afferents (CSD1 and CSD2) with motorneurones of the four proximal muscle groups in the 5th walking legs of crayfish (Procambarus clarkii, Pacifastacus leniusculus) have been studied in an in vitro preparation. Reflex responses to mechanical stimulation of the CSDs were studied in single neurones by means of intracellular techniques. Within each motorneurone pool, both excitatory and inhibitory reflex responses occurred, although sometimes no reflex connections were found. When present, they could be classified into 'levation' and 'depression' reflexes, corresponding to negative and positive feedback effects respectively. Each motorneurone receives input from a number of different CSD afferents (mean values between 3.0 and 5.8). Using electrophysiological and pharmacological tests, it was demonstrated that at least 32% of all connections were monosynaptic. In preparations showing fictive locomotion, phasic CSD stimulation was shown to be able to entrain anterior levator and depressor motorneurone activity in 95% of cases. The results thus demonstrate the importance of sensory feedback from the CSDs in shaping the final motor output.

Functional aspects of central electrical coupling in mechanoreceptor afferents of crayfish.

Cuticular stress detector afferents (CSD1 and CSD2) in the walking legs of crayfish have been studied in an in vitro preparation allowing intracellular recordings of single neurones during mechanical stimulation of the organs. Lucifer yellow staining of single fibres showed them to be dye-coupled through junctions presumably located near their point of entry into the ganglion. In intracellular recordings from identified sensory fibres with 30-80-mV impulses, coupled afferent spikes were seen as depolarizing potentials of short duration but smaller amplitude. Coupling sometimes involved fibres with quite different response characteristics to mechanical stimuli. Coupled spikes summated during strong mechanical stimuli. In silent CSD fibres, antidromic spikes could occur which were triggered by the depolarization due to coupling. Antidromic spikes were more frequent during strong stimuli and were in some cases shown to be triggered in a cooperative way when two afferent spikes synchronously reached the ganglion. Our results suggest that central coupling of primary afferents may be a widespread phenomenon in Crustacea and that it provides a threshold mechanism for selective amplification of strong stimuli.

Dark adaptation of toad rod photoreceptors following small bleaches.

The recovery of toad rod photoreceptors, following exposure to intense lights that bleached 0.02-3% of the rhodopsin, has been investigated using the suction pipette technique. The post-bleach period was accompanied by reduced flash sensitivity, accelerated kinetics, and spontaneous fluctuations (noise). The power spectrum of the fluctuations had substantially the form expected for the random occurrence of single-photon events, and the noise could therefore be expressed as a "photon-noise equivalent intensity". From the level of desensitization at any time, the after-effect of the bleach could also be expressed in terms of a "desensitization-equivalent intensity", and this was found to be at least a factor of 20 times higher than the noise-equivalent intensity at the corresponding time. Our results indicate that a bleach induces two closely-related phenomena: (a) a process indistinguishable from the effect of real light, and (b) another process which desensitizes and accelerates the response in the same way that light does, but without causing photon-like noise. We propose a mechanism underlying these processes.