GéS.F. Ruigt

Lysophosphatidic acid induces inward currents in Xenopuslaevis oocytes; evidence for an extracellular site of action

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that can elicit platelet aggregation, smooth muscle contraction and, in fibroblasts, cell proliferation. We now report that LPA in nanomolar concentrations evokes an inward current in native, defolliculated Xenopus laevis oocytes. Extracellular application of LPA from a pressure pipette to the surface of the oocyte induced an immediate response. In contrast, intracellular injection of the same amount of LPA failed to elicit a response. These data suggest the existence of a Ca(2+)-mobilizing, endogenous LPA receptor in the Xenopus laevis oocyte cell membrane.

A large scale, high resolution, automated system for rat sleep staging. I. Methodology and technical aspects

An automatic rat sleep classification system is described which records and analyses bioelectrical signals from 32 rats over extended periods of time. At present this system is used routinely for the screening of drug effects on sleep. The analysis is based on 3 signals, the parieto-occipital EEG, nuchal EMG and a movement indicator signal. The on-line analysis is done per epoch of 2 sec and involves power spectral analysis of the EEG and rectification and integration of the EMG and movement signals. The automatic sleep staging into 6 stages (active and quiet waking; quiet, deep, pre-REM and REM sleep) is performed off-line. Parameters derived from a discriminant analysis of visually scored tracings of individual rats constitute the basis for the automatic scoring procedure. The movement index is used to discriminate between active and quiet waking, while the use of the EMG level improves the separation of waking and REM sleep. After the construction of hypnograms from these computer scorings a set of parameters can be extracted which characterizes the sleep-waking behavior of each individual rat. These parameters are then used to compare statistically the 2-4 treatment groups which make up each experiment of 32 rats. Experimental validation of the system is reported in an accompanying paper.

A large scale, high resolution, automated system for rat sleep staging. II: Validation and application

An automated rat sleep staging system was used to describe sleep-waking behaviour in a large number of rats. The computer scorings were validated by visual analysis of a limited set of data by two human raters and the agreement varied between 82 and 100% for homogeneous segments of the different sleep-waking stages. The automated system proved to be very consistent in view of the small variation in placebo values over 110 experiments. The data show that classification of rat sleep waking behaviour into 6 different stages is both advisable and feasible. The experiments further show that rat age (over a range from 0.5 to 2 years) does not greatly affect rat sleep-waking behaviour.

SR 57746A attenuates cytostatic drug-induced reduction of neurite outgrowth in co-cultures of rat dorsal root ganglia and Schwann cells

A co-culture system of intact rat dorsal root ganglia (DRG) with Schwann cells was used to evaluate the potential neurotrophic activity of SR 57746A. Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of SR 57746A. Neurite outgrowth of intact DRG was increased by SR 57746A and this was more obvious in the presence of co-cultured Schwann cells. The neuroprotective properties of SR 57746A were studied in co-cultures of DRG and Schwann cells, in which neuritogenesis was reduced by the cytostatic drugs cisplatin, vincristine and taxol. It was found that neurite outgrowth from DRG treated with cisplatin (3 micrograms/ml) and 10 microM SR 57746A for 3 days was significantly higher than after treatment with cisplatin alone. Similarly, neuritogenesis from DRG treated with taxol (0.01 microgram/ml) or vincristine (0.5 ng/ml) in combination with 10 microM SR 57746A was significantly increased compared to treatment with taxol or vincristine alone. When intact DRG were incubated in vitro with 3 micrograms/ml cisplatin and without Schwann cells, 10 microM SR 57746A also had a neuroprotective effect. These data suggest that SR 57746A has neuroprotective potential and that this effect does not depend solely on the presence of Schwann cells.

The endegenous muscarinic acetylcholine receptor in Xenopus oocytes is of the M3 subtype

The muscarinic acetylcholine receptor of the Xenopus oocyte was characterized electrophysiologically. Iontophoretic responses to acetylcholine were inhibited by the muscarinic antagonists 4-DAMP, pirenzepine and AF-DX 116 at respective IC50 values of 7 nM, 15 microM and 2 microM. This antagonist sensitivity order indicates that the receptor is of the M3 muscarinic subtype.

Synthesis of sialic acid-lipid conjugates and their neuritogenic effects on N1E.115 neuroblastoma cells☆

Several sialic acid-lipid conjugates having one or more sialic acid residues were prepared via phosphite and methylthiomethyl intermediates. The synthetic compounds were found to promote neurite outgrowth on N1E.115 neuroblastoma cells. In particular, the trisialocholesterol derivative 19 exhibited potent neuritogenic activity.

α-Sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of laminin

Schwann cells play an important role in peripheral nerve regeneration. Here, we report the effect of alpha-sialyl cholesterol (alpha-SC), a derivative of the sialic acid-containing natural gangliosides, and the cytostatic agents, cisplatin, taxol and vincristine on the laminin production in Schwann cell cultures isolated from rat sciatic nerves. Laminin, one of several extracellular matrix components produced by Schwann cells, is known to potentiate axonal outgrowth. Laminin content was increased by alpha-SC, starting at 7.0 micrograms/ml with a maximal effect at 22.4 micrograms/ml (30%, P < 0.001). The three cytostatic drugs, dose-dependently reduced laminin content in Schwann cell cultures: (1) cisplatin at a threshold dose of 2 micrograms/ml (-26.4%, P < 0.001); (2) taxol, starting at a dose of 1 ng/ml (-8.0%, P < 0.05); and (3) vincristine, starting at 0.5 ng/ml (-5.9%, P < 0.05). Cultured Schwann cells were incubated with cytostatic drugs in combination with increasing amounts of alpha-SC and it was found that, depending on the cytostatic drug concentration used, alpha-SC could reduce or completely prevent the cytostatic drug-induced reduction of laminin in Schwann cell cultures. Co-treatment with alpha-SC also reduced part of the morphological changes caused by the cytostatic drugs. alpha-SC did not counteract the anti-proliferative effect of the cytostatic drugs on K-562 human erythroleukemia cells. In conclusion, alpha-SC increased laminin content in Schwann cell cultures and protected Schwann cell cultures against the decrease of laminin by cytostatic drugs without interfering with the anti-proliferative potential, suggesting that alpha-SC may have clinical use in protecting cancer patients against the neurotoxic effects of cytostatic drugs.

Morphometric analysis of cisplatin-induced neurite outgrowth in N1E-115 neuroblastoma cells

Cisplatin, a widely used cytostatic drug for the control of a variety of neoplastic tumors, unexpectedly induced neurite outgrowth in N1E-115 neuroblastoma cells and this phenomenon was studied further in detail with morphometric analysis. As expected, cisplatin dose-dependently reduced cell number. At the same time, however, cisplatin affected the morphology of the neuroblastoma cells that changed from small rounded cell bodies into large flat cell bodies with neurites. The neurite length/cell as a function of cisplatin concentration showed a bell-shaped curve. The maximal effect (1200% of control) on neurite length/cell was observed at 1 microgram/ml cisplatin. In conclusion, cisplatin induced cellular differentiation in N1E-115 neuroblastoma cells at and just above threshold doses for cytostatic activity.