Effects Of Adenosine On The Intracellular Ca2+ Oscillations And High Voltage-activated Calcium Channel Current In Rat Hippocampal Neurons

In order to afford the experiment basis for the adenosine-induced antiepileptic effect, effects of adenosine (Ade) on high voltage-activated calcium channel current (IHVA) and Ca²⁺ oscillations were studied in the cultural hippocampal neuronal cells of the 24-hour SD rats.Methods:The 24-hour SD rats were taken as material objects; the hippocampal neuron primary culture systems were established by digesting of pancreatic enzyme and blowing of dropping pipette in the condition culture medium. The actions of Ade on the IHVA and the Ca²⁺ oscillations were investigated respectively by laser confocal microscope and whole-cell patch clamp technique. Ade was applied by extracellular bath, and its effect was determined by comparing the amplitude of IHVA, the frequency and amplitude of Ca²⁺ oscillations before and after Ade application.Results:The hippocampal neurons were used when they had shaped network after 7-9 days of culture. Loaded with probe Fluo-3/AM and imaged with a laser scanning confocal microscope in a bath, approximately 32% of them exhibited the intracellular spontaneous and synchronous Ca²⁺ oscillations:â‘´Calcium oscillations were depressed in a dose dependent manner. Ade inhibited the frequency and amplitude of the spontaneous Ca²⁺ oscillations respectively from (1.2±0.2)/min (0.02±0.003HZ), (1.87±0.17) to (0.3±0.05)/ min (0.005±0.001HZ), (1.0±0.07). When induced by 60mmol/L KCl, the frequency and amplitude of calcium oscillations showed a marked enhancement, however, they were also depressed after sequential application of Ade: from(2.39±0.22)/min(0.04±0.003HZ) ,(2.45±0.27) to (0.44±0.13)/min(0.01±0.002HZ), (1.12±0.08).⑵Slow inactivating inward currents were elicited in response to a series of depolarizing voltage values. Then it was confirmed by patch clamp technique that bath application of Ade at concentrations of 0.1,1,10 and 50umol/L could inhibit IHVA dose-dependently by(5.2±2.3) %(n=6,P>0.05),(8.1±1.9) %(n=6,P>0.05),(28.4±2.8)%(n=6,p<0.05),(43.5±4.3) % (n=6,p<0.05), comparing with control group .The I-V curves shifted upwardly, but Ade had no effect on the maximum potential, the reversal potential and the threshold potential for activating IHVA. A kind of L-type calcium channel antagonists :nifedipine could depress the reduce of IHVA by Ade.Conclusion:Ade could inhibit IHVA, the frequency and amplitude of Ca2+ oscillations in rat hippocampal neuronal cells. It may be involved in the mechanism of endogenous adenosine with respect to the antiepileptic effect.