Human IGR1 cells are a model for malignant melanoma. Since progression through the cell cycle is accompanied by transient cell hyperpolarization, we studied the properties of potassium and chloride ion channels and their impact on cell growth. The major potassium current components were mediated by outward rectifying ether à go-go (hEAG) channels and Ca2+-activated channels (KCa) of the IK/SK type. The major chloride channel component was activated by osmotic cell swelling (Clvol). To infer about the contribution of these channels to proliferation, specific inhibitors are required. Since there is no specific blocker for hEAG available, we used the tricyclic antidepressant imipramine, which blocked all channels mentioned, in combination with blockers for KCa (charybdotoxin) and Clvol (DIDS and pamoic acid). Incubation of IGR1 cells for 48 hr in 10-15 mM imipramine reduced DNA synthesis and metabolism without significant effects on apoptosis. hEAG channels were most sensitive to imipramine (IC50: 3.4 microM at +50 mV), followed by KCa (13.8 microM at +50 mV) and Clvol (12 microM at -100 mV), indicating that hEAG expression may be of importance for proliferation of melanoma cells. The contribution of KCa channels could be excluded, as 500 nM charybdotoxin, which completely blocked KCa, had no effect on proliferation. The impact of Clvol also seems to be minor, because 500 microM pamoic acid, which completely blocked Clvol, did not affect proliferation either.

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community. The reproduction of naturally occurring triploids has been reported to occur only through parthenogenesis, gynogenesis or hybridogenesis. The bisexual reproduction of pure triploids has been considered to be impossible because of the problem of equally distributing three chromosome sets in meiosis. Here we report geographically isolated populations of green toads (Bufo viridis complex) that are all-triploid and reproduce bisexually.

No abstract

The bis-barbituric acid oxonol, DiBAC 4 (3) is used as a standard potentiometric probe in human cells. However, its fluorescence depends not only on membrane potential but also varies with nonpotential related changes in the amount of intracellular free and bound dye. This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC 4 (3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC 4 (3), the logarithmic relation between cell fluorescence and dye concentration turned into a virtually linear function intersecting with zero. Consequently, calibration can then be confined to determination of the fluorescence of depolarized cells stained with the same concentration as used for the actual measurement of membrane potential. Unexpectedly, quenching of fluorescence occurred in totally depolarized cells at concentrations higher than 6,250 nM. Linearity and quenching could be confirmed by additional experiments on Chinese hamster ovary CHO-K1 and B lymphoblastoid LCL-HO cells. ' International Society for Advancement of CytometryKey terms bis-barbituric acid oxonol; flow cytometry; membrane potential; mV-scale calibration PATCH-clamp recording is considered to be the reference method to quantitatively measure the plasma membrane potential V p in human cells. A disadvantage in respect of routine use is that this method is time-consuming and laborious thus usually resulting in measuring values for only a small number of cells (1,2). Therefore, voltage-sensitive anionic and cationic dyes, which partition between the extracellular staining buffer and the cytoplasm under the influence of V p according to a Nernstian distribution, have been applied as an alternative (3-12).Due to their strong accumulation in mitochondria (13,14), cationic dyes report V p alone only when the mitochondrial membrane potential V m remains constant during the measurement. To ensure this condition, V m was specifically abolished by ionophores in some studies (15)(16)(17). With increasing concentration, the monomer form of cationic dyes, among them carbocyanines as the most popular dye class, is usually more and more displaced by dimers or higher aggregates. This process takes place both with dye dissolved in aqueous solution or bound to cell compartments, and is accompanied by distinct spectral shifts in absorbance and fluorescence maxima leading to quenching of the fluorescence of the monomers (18-21). For example, DiSC 3 (5) monomers show red flu...

In the present study, the distribution of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA)-receptor type was immunohistochemically demonstrated in healthy human skin (n = 22) and healthy buccal mucosa (n = 20). Moreover, the intracellular calcium concentration of HaCaT-cells and native human keratinocytes were studied under the influence of the selective agonist NMDA and the selective NMDA-antagonist MK-801. Immunohistochemical imaging of NMDA receptors in healthy epidermis showed a positive reaction in the stratum basale, spinosum and granulosum, whereby the greatest expression was observed in the granular layer. In the mucosal preparations, the distribution of NMDA receptors was observed to be equal in all cell layers. In the cell culture (HaCaT-cells), NMDA concentrations between 25 microM and 1 mM resulted in a significant increase in the number of cells showing elevated intracellular calcium concentration. This effect could be significantly reduced by prior application of MK-801 (100 micro M). In supplementary tests on HaCaT-keratinocytes, blockade of the keratinocytic NMDA receptors with MK-801 suppressed the differentiation of the cells (expression of cytokeratin 10). The proliferation of cells was not influenced by NMDA. The investigations showed that glutamate receptors of the NMDA type have an influence on keratinocytic calcium concentration. This appears especially important for the differentiation of keratinocytes.