Further study was undertaken to define the character of functional interplay between NAD+ availability and changes in the specific binding of NAD+ by brain synaptic membranes in STZ-diabetic rats (70 mg/kg b.w.) which were treated by single injection for 6 hours (short-term) or chronically for two weeks (long-term) after 6 or 4 weeks of diabetes respectively with nicotinamide (NAm) or nicotinoyl-GABA (N-GABA) in low or high (20 and 200 mg/kg b.w.) doses. The brain NAD+ and ATP contents were significantly reduced in diabetes vs. control (respectively by 27.3±1.9 % and 30.1±2.7 %). No difference occurred between increments in NAD+ contents after single administration of high NAm dose in control and diabetes indicating preserved nervous cells ability to synthesize NAD+ in sufficient amounts. Depletion of brain NAD+ and ATP contents defined in diabetes could be regarded as a consequence of free radical- and oxidant-induced increase in ADP-ribosylation since poly(ADP-ribose)polymerase activation was currently established. Diabetes was also found to be associated with enhanced oxidative stress in brain as it is evident from 2.9–fold increase in accumulation of thiobarbituric acid reactive substances vs. control. These changes were accompanied by 51.0 % increase in maximal capacity of [U-14C]NAD+ binding sites (Bmax) of synaptic membranes. In spite of augmented binding, NAD+ (1 mmol/L) failed to evoke the release of serotonin from the synaptosomes isolated from diabetic rat brain to the level of control. While NAD+ and ATP contents were similarly counteracted by low and high doses of NAm and N-GABA treatments, NAD+ reception fully restored only in case of chronic administration of high Nam dose. Discrepancies in efficacy of the drugs to restore NAD+ contents and normalize NAD+ binding and serotonin release after long-term treatments corresponded to their efficacy as free radicals scavengers and Parp inhibitors being the strongest in case of high NAm dose.
Our data provide further evidence that diabetes may cause physiologically drastic failure in realization of serotonin-mediated effects in CNS.It was found that striking increase in spontaneous and evoked serotonin release associated with diabetes is, at least partially, dependent on Gs protein/cAMP/PKA-mediated facilitatory pathway in synaptic endings related to enhanced mono-ADP-ribosylation of extranuclear proteins. NAm treatment, initiated after diabetes duration of 4 weeks, virtually normalized both mono-ADP-ribosylation and serotonin release.
In conclusion, the present study confirms the complex pattern of vitamin PP neuroprotective action used in high therapeutic doses. Its beneficial effects on diabetes-associated brain failures can be related to normalization of NAD+ reception most likely due to its antioxidant and anti-ADP-ribosylating properties.
Key words: nicotinamide, nicotinoyl-GABA,NAD-binding protein, mono- and poly-ADP-ribosylation,protein kinases А/С, Na+,K+-АТPase, serotonin, uptake, release, synaptosomes, diabetes mellitus.
СПИСОК СКОРОЧЕНЬ
ГАМК – г-аміномасляна кислота;
N-ГАМК – нікотиноїл-ГАМК;
ТБК-активні продукти – продукти, які реагують з тіобарбітуровою кислотою;
ЦД1 - цукровий діабет 1 типу;
4-AP - 4-амінопіридин;
СТХ - холерний токсин;
G-білки - GTP-зв’язувальні білки;
LTX - латротоксин
NАm - нікотинамід;
Parp - [Poly(ADP-ribose) polymerase] - полі-ADP-рибозилполімераза;
PKA - протеїнкіназа А;
РКС - протеїнкіназа С;
ТТХ - тетродотокcин;
VTD - вератридин;