Wolfgang Probst

Modulatory effects of different temperatures and Ca2+ concentrations on gangliosides and phospholipids in monolayers at air/water interfaces and their possible functional role

Summary1.Gangliosides are neuraminic acid-containing glycolipids preferently localized in nervous membranes and showing physicochemical peculiarities, e.g., drastically changing amphiphilic properties by Ca2+ binding. On account of this they are favorite compounds to act as modulators of membraneous organization and functions during synaptic transmission. Lipid monolayers are suitable experimental systems for the study of the surface behavior of amphipatic molecules and therefore are useful to interpret membraneous organization.2.The surface pressure/area isotherms of monolayers of different individual gangliosides (GM1, GD1a, GD1b, GT1b) of an artificial reconstituted and a natural ganglioside mixture from bovine brain and of ganglioside mixtures from different brain parts of summer- and winter-adapted dsungarian hamsters were compared at three temperatures (11, 20, and 37°C) with egg phosphatidylcholine (PC) and phosphatidylserine (PS) monolayers. The monolayers were formed in a Teflon trough on a triethanolamine/HCl-buffered (pH 7.4) subphase, in some cases containing different amounts of CaCl2.3.The surface pressure/area isotherms of ganglioside monolayers, in contrast to phospholipids, generally showed slowly rising slopes, with transitions from the liquidexpanded to the liquid-condensed state at a surface pressure of 20–30 mN/m. Ganglioside monolayers, in particular from GD1a or GT1b versus GD1b or from mixtures from summer- versus winter-adapted hamster brain, were differently affected by temperature and/or by Ca2+. PS monolayers were slightly condensed only by Ca2+. PC monolayers, however, were influenced neither by temperature nor by Ca2+. In mixed monolayers of the unpolar natural lipid cholesterol (Ch) and the disialoganglioside GD1a, intermolecular interactions were indicated.4.Ganglioside monolayers, in contrast to phospholipids, were shown to be easily modulated by temperature and/or Ca2+ ions, thus enabling gangliosides to act as possible membrane modulators, e.g., during synaptic transmission. In particular, the differences concerning the influences of temperature and/or Ca2+ on the surface behavior of ganglioside mixtures from the brain of summer- compared with winteradapted hamsters are correlated with other physiologically relevant data.

Influence of temperature changes on the ability of gangliosides to complex with Ca2

Abstract 1. 1.|The influence of temperature changes on Ca 2+ -binding to brain ganglioside mixtures of different polarity, to single gangliosides (G M1 , G D1a , G T1b ) and to their deceramide was investigated potentiometrically by means of ion-selective electrodes. 2. 2.|Following cooling (3.5°C/min.) from 37 to 13°C the Ca 2+ -binding to gangliosides, except G M1 , was increased (7–30%). 3. 3.|Subsequent rewarming from 13 to 37°C resulted in up to 100% release of previously-bound Ca 2+ . 4. 4.|When comparing the maximal absolute binding difference of Ca 2+ to gangliosides during temperature changes a decrease of these differences could be stated which corresponds to an increase in the polarity of the gangliosides. 5. 5.|From these experiments it is concluded that a higher polarity of neuronal gangliosides is responsible for a lower thermal sensitivity of Ca 2+ -binding to these compounds. This may be involved in the process of thermal adaptation of ectothermic vertebrates.

In-vitro-studies on the influence of cations, neurotransmitters and tubocurarine on calcium-ganglioside-interactions.

Abstract The influence of K + , Na + , Mg ++ , Li + , a serotonin, acetylcholine and tubocurarine on calcium-ganglioside-interactions was studied by way of equilibrium dialysis using 45 Ca as tracer. Experiments were carried out at 22 °C and 4 °C, respectively. The concentrations of the substances were in the range of physiologically relevant conditions. Cations caused a release of Ca ++ from calcium-ganglioside-complexes in the sequence of their molar efficiency: Mg ++ ≈ Li + > K + ≈ Na + . Tubocurarine, serotonin and acetylcholine also affected calcium-ganglioside-interactions. Ca ++ was displaced from ganglioside most effectively by tubocurarine, followed by serotonin, whereas acetylcholine competed considerably more weakly.

Method for the OsO4-free preservation of brain tissue suitable for immunocytochemistry in TEM.

Following conventional OsO4-fixation and embedding of brain tissue in Epoxide resins for ultrastructural investigations, on the one hand, the wellknown good depiction of structures is obtained, on the other hand, however, according to these procedures a considerable loss up to 80% of glycolipid-bound and up to 50% of glycoprotein-bound neuraminic acid of important native compounds has to be put up with. The fixation of brain tissue by 1% glutaraldehyde and 1% paraformaldehyde in 0.1 mol/l Na-K-phosphate-buffer, followed by dehydration in acetone or ethanol at--25 degrees C and embedding in Lowicryl K4M preserves for instance amphiphilic glycolipids (gangliosides) to a high degree (95%) and in addition provides a good depiction of neuronal structures. The incubation of ultrathin sections of carp brain, treated as described above, with the specific antibody Q211 against polar gangliosides gave first evidence for a specific labelling of these amphiphilic glycosphingolipids and their localization within the brain tissue.

Influence of Ca 2+ and Temperature Changes on the Surface Requirement of Gangliosides and Phospholipids in Monolayers

The surface pressure in monolayers of individual gangliosides (GT1b, GD1b, GD1a, GM1, lyso-GM1), sulphatide, ceramide, phospholipids (PS, PC) artificial mixtures of peptides and gangliosides and of natural ganglioside mixtures from bovine and ice- fish brain were investigated by means of a Wilhelmy balance in a rectangular teflon trough. The surface pressure/area isotherms were compared at 11°, 20° and 37° C and different Ca2+-concentrations in the subphase (0; 0.01 and 0.5 mM CaCl2).

Ultrastructural Localization of Calcium at Synapses and Modulatory Interactions with Gangliosides

In 1975/76 we published the functional hypothesis of an involvement of sialoglycomacromolecules, especially gangliosides, in the process of synaptic transmission, including memory formation (Rahmann, 1976; Rahmann et al., 1975; 1976). In our proposal it had been discussed that due to the ability of ganglioside-bound negatively charged sialic acids to form labile complexes together with Ca2+-ions, these glycosphingolipids are assumed to act as modulatory compounds for the Ca2+-dependent release of transmitter substances at synapses. Since 1975 we have been able to add to this hypothesis extensive experimental evidence (Rahmann, 1983; 1984; Rahmann et al., 1982; Probst et al., 1984). In the meantime it also had been supported by several other authors (Svennerholm, 1980; Tettamanti et al., 1980; Maggio et al., 1981; Veh and Sander, 1981; Leskawa and Rosenberg, 1981). The essential basis for our model is the well-known fact that almost every stage of neuronal activity, especially electrical responsiveness, depends on the presence of extra-cellular Ca2+. With regard to this, during recent years extensive experimental efforts were undertaken to establish subcellular Ca2+-deposits in the synaptic terminal. Rough endoplasmic reticulum, mitochondria and vesicles were determined as intracellular Ca2+-storage structures (McGraw et al., 1980; Chan et al., 1983). The electronmicroscopical methods used until now, however, failed to establish extra-cellular deposits of Ca2+.

Involvement of temperature in the composition of fish brain gangliosides

Abstract 1. 1.|The concentration and composition of the brain gangliosides of 35 fish species belonging to the agnathes, chondrichthyes and osteichthyes living in biotops of different temperature were analysed. 2. 2.|The concentration varies between values of 110–750 μg NeuAc/g fresh wt. 3. 3.|The composition of gangliosides deviates considerably with the following tendency: the lower the environmental temperature–the higher the degree of sialylation (⋍ polarity) of brain gangliosides. 4. 4.|By means of equilibrium dialysis using 45 Ca 2+ a significant thermosensitivity of Ca 2+ -ganglioside complexes was shown only when the ganglioside molecules were inserted into the lipid bilayer of liposomes.