Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology

Platelets are unique cells of human body: they lack nucleus, are rather small in size (1–2 μm) and involved in several physiological functions, including hemostasis, immunity and angiogenesis. Platelets play a key role in the initiation of thrombosis upon injury of the blood vessels of the arterial bed, in which blood flows with high shear rates are observed. According to the generally accepted concepts, the reaction of platelets to endothelial injury at local shear rates of more than 1000 s^–1 is the primary binding of the GPIb-IX-V receptor complex glycoproteins with von Willebrand factor, a large multimeric blood protein which can specifically bind to collagen fibers. For further performance of their functions, and first of all, for stable attachment to the injured surface, platelet has to be activated. There are more than ten types of receptors on the platelet membrane, which trigger several cascades of intracellular signaling that leads to the restructuring of the cytoskeleton, granule secretion and activation of integrins, which provide the ability of platelets to strong adhesion and aggregation. This review is focused on the biophysical aspects of the interaction of transmembrane glycoproteins and integrins with extracellular ligands, as well as modern ideas about the mechanisms of platelet activation that is necessary to stabilize their primary adhesion and aggregation.

In contrast to frog neuromuscular synapses, where noradrenaline (norepinephrine) and its analogues caused synchronization of the acetylcholine release process, in mouse diaphragm endplates noradrenaline increased the degree of asynchrony of neurosecretion. The effect of noradrenaline on release timing persisted at different levels of external calcium ions (0.25–2.0 mM) and was abolished in presence of both α- and β‑adrenoblockers phentolamine and propranolol. The computer reconstruction of multiquantal endplate currents accounting for experimentally observed modification of release kinetics under noradrenaline showed that the rise time of postsynaptic response changes to a greater extent than the amplitude and falling phase of the multiquantal responses. We conclude that there exists a principal difference in the action of noradrenaline in the cholinergic neuromuscular synapses of warm-blooded and cold-blooded animals that can be accounted for by the differences in the type of adrenoreceptors involved in the modulation of synaptic transmission and/or in the involvement of distinct intracellular pathways triggered by receptor activation.

Binding of potassium ions through an access channel from the cytoplasmic side of Na⁺,K⁺-ATPase and the effect of pH and magnesium ions on this process have been studied. The studies were carried out by a previously developed method of measuring small increments of the admittance (capacitance and conductivity) of a compound membrane consisting of a bilayer lipid membrane with adsorbed membranes fragments containing Na⁺,K⁺-ATPase. The capacitance change of the membrane with the Na⁺,K⁺-ATPase was induced abruptly by release of protons from a bound form (caged H⁺) upon a UV-light flash in the absence of magnesium ions. The change of admittance consisted of an initial fast jump and a slow relaxation to a stationary value within a time of about 1–2 s. The kinetics of the capacitance relaxation depended on pH and the concentration of magnesium and potassium ions. The dependence of the rapid capacitance jump on the potassium concentration corresponded to the predictions of the model developed earlier that describes binding of sodium or potassium ions in competition with protons. The effect of magnesium ions can be explained by the assuming that they bind to the Na⁺,K⁺-ATPase and affect binding of potassium ions because of either changes in protein conformation or the creation of an electrostatic field in the access channel on the cytoplasmic side.

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca²⁺ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca²⁺ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca²⁺ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca²⁺ overload, while only a small portion of neurons were able to compensate the intracellular Ca²⁺ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φ_mit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φ_mit so that an uncoupling agent FCCP did not cause any additional changes in φ_mit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φ_mit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.

The involvement of endocytosis in the Na⁺ ion uptake from the external medium by the cells of suspension culture derived from A. thaliana (Col-0) leaves was investigated. Na⁺ ion uptake by endocytic structures occurred following the addition of NaCl at the final concentration of 100 mM to the incubation medium. The presence of Na⁺ in membranous structures was recorded using fluorescence microscopy by colocalization of FM4-64, a marker of endocytosis structures, and Asante NaTRIUM Green-2 TMA⁺ salt (ANG-2 TMA), a membrane impermeable probe for sodium ions, that enabled the detection of Na⁺ absorbed by the cells via endocytosis but not through ion channels or transporters of the plasma membrane. Following a 1.5-h incubation of the cells in the presence of NaCl, FM4-64 and ANG-2 TMA, fluorescence of the probes was colocalized in structures with sizes ranging from 800 to 3000 nm. It was shown by electron microscopy that NaCl added to the cell incubation medium stimulated vesiculation and vacuolization of the cytoplasm, formation of plasma membrane invaginations, as well as fusion of microvacuoles with each other. The size of the structures, in which the colocalization of the two probes was detected by fluorescent microscopy, matched the size of the microvacuoles revealed by the electron microscopy. The obtained results indicate the capture of sodium ions contained in the apoplast by endocytosis invaginations, their subsequent internalization by the cells, and transfer into microvacuoles.

The analysis of neuronal viability of cerebellar neurons in primary culture during long-lasting (240 min) treatment with glutamate revealed that forskolin (1 μM), an adenylate cyclase activator, prevents apoptosis and necrosis of cells, thus exhibiting neuroprotective properties. As the cytotoxic action of glutamate causes an increase of the intracellular Ca²⁺ concentration, we further studied forskolin influence on glutamate-evoked Ca²⁺ responses of neurons. A short-time pretreatment with 1 μM forskolin significantly decreases Ca²⁺ entry into neurons during glutamate action. The obtained results demonstrate an important role of adenylate cyclase and cAMP in regulation of the Ca²⁺ entry into neurons and intracellular signaling pathways preventing neuronal death of cerebellar neurons in excytotoxic stress.