Studies devoted to various neurodegenerative disorders pointed out to malfunction of certain isoforms of the Na,K-ATPase in the brain. Due to the presence of 3 various isoforms of the catalytic α-subunit in the brain tissue the data obtained by our investigation of enzyme kinetics represent the cumulative activity of all 3 isoforms of the enzyme. Previously it was documented that administration of TMT to rats induced neurodegeneration . By in vitro studies it was shown that TMT was able to inhibit the Na,K-ATPase in synaptosomes from the forebrain of mice  indicating that in neurotoxic action of trimethyltin (TMT) this enzyme might be involved. In the present study we tried to broaden the information concerning the molecular principles of expected alterations of Na,K-ATPase in vivo in rats, four weeks after administration of TMT. Our investigations using measurements of enzyme kinetics showing higher Na,K-ATPase activities in the lower concentration range of ATP indicate better utilization of the energy substrate confirmed also by lowered value of Km in TMT-treated non-handled animals when compared to non-handled healthy controls. The number of active Na,K-ATPase molecules remained unchanged as indicated by similar values of Vmax in TMT and the control group, both without handling. So the effect of TMT on the Na,K-ATPase from frontal cortex seems to be different when measured in vitro and in vivo. Our measurement four weeks after in vivo administration of TMT showed higher effect in the presence of physiologically relevant concentrations of ATP (0.16–0.80 mmol·l− 1), while the in vitro measurements of Costa  were done at higher concentration (2 mmol·l− 1). Another important fact probably contributing to the difference between in vivo and in vitro experiments is the probably reduced presence of TMT due to urinary excretion of TMT during the in vivo experiment. Previously, it was documented that after 8 days the TMT presence decreased almost by 50% in mice and rats .
Previous study of TMT-induced neurodegeneration showed that handling of young adult male rats improved the spatial memory in healthy and also in TMT-treated rats and prevented the memory impairment induced by trimethyltin . Therefore the second aim of the present study was oriented to the effect of handling of rats on the Na,K-ATPase properties in healthy rats and also in rats with TMT-induced neurodegeneration.
Our present results of enzyme kinetic investigations indicate that the Na,K-FATPase might be involved in positive effects of handling in healthy rats as well as in rats with TMT-induced neurodegeneration. While in both groups of handled rats the number of active enzyme molecules remained unchanged, as indicated by unaltered Vmax value when comparing to respective controls, the ability of the enzyme to bind and utilize the energy substrate ATP was markedly improved as indicated by lowered Km values. So, the handling of rats may affect similarly the energy utilization and extrusion of superfluous sodium from cells in frontal cortex in healthy rats and in rats with TMT-induced neurodegeneration. The positive impact of enhanced Na,K-ATPase functionality in the brain tissue probably plays an important role in improvement of spatial memory in healthy non-treated rats and rats with TMT-induced neurodegeneration when handled for 6 weeks. The importance of Na,K-ATPase in prevention of neurodegeneration was supported also in the experimental model of epilepsy where stimulation of Na,K-ATPase with specific antibody (DRRSAb) restored the crossing activity of pilocarpine-treated mice in the open-field test .
This study provides also new information concerning the expression of individual α subunits of Na,K-ATPase (α1, α2, α3) in the frontal cerebral cortex in consequence of handling of healthy rats or rats with TMT-induced neurodegeneration. The global maintenance of intracellular sodium homeostasis was probably not affected by TMT-treatment of adult rats in handled as well as in non-handled rats as indicated by unaltered expression of α1 subunit. This ubiquitously expressed isoform is responsible for global maintenance of intracellular sodium homeostasis [29, 30].
The increased expression of glial isoform α2 in handled rats with TMT-induced neurodegeneration seems to be interesting in view of pathological alterations in consequence of poisoning by TMT. It was previously documented that malfunction of this isoform of Na,K-ATPase is probably involved in development of familial hemiplegic migraine type 2 . However, the increased expression of glial isoform α2 in TMT-affected and handled rats may represent an interesting effect requiring further investigations for explaining the physiological relevance of this fact.
Based on our analysis of the protein expression of Na,K-ATPase α3 subunit it may by hypothesized that increased expression of this subunit could play a role in the neuroprotective mechanism of handling in healthy rats whose were not poisoned by TMT. Previously it was documented that α3 subunit is exclusively specific for fully differentiated neurons , thus we can suppose that handling of healthy animals might act at the neuronal level. Na,K-ATPase stimulates the growth of dendrites during the development of the brain where signal growth is triggered by signal transduction and probably plays a role in neurogenesis . Malfunction of α3 is probably involved in development of several neurological disorders, as it was suggested also for rapid-onset dystonia parkinsonism in mice . So, it may be hypothesized that increased expression of neuronal α3 probably is involved in the mechanism of previously reported improvement in memory of handled control animals . This hypothesis seems to be in agreement with previous observation of altered spatial learning, motor activity and anxiety in α3-deficient mice . In addition, the animals with the haploinsuficiency gene for this subunit of Na,K-ATPase were accompanied by cognitive deficits .