Effect of the Organic Calcium Antagonist D-600 on Cerebrocortical Vascular and Redox Responses Evoked by Adenosine, Anoxia, and Epilepsy

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Abstract
The purpose of this study was to investigate the role of calcium ions in cerebrocortical vasodilatation and oxidized and reduced nicotinamide adenine dinucleotide (NAD/NADH) redox responses evoked by adenosine, anoxia, and epileptic seizures. The brain cortex of chloralose-anaesthetized cats was treated locally with gallopamil-hydrochloride (D-600) and verapamil (Isoptin®). These organic calcium antagonists decrease the inward movement of calcium ions into vascular smooth muscle cells. Cerebrocortical vascular volume (CVV) and NADH fluorescence were measured in vivo by fluororeflectometry. Adenosine and calcium antagonists were dissolved in artificial cerebrospinal fluid (mock CSF) and applied topically to the brain cortex by superfusion. Adenosine (10−8 to 10−3 M) resulted in concentration-dependent increases in CVV. The NAD/NADH redox state was not altered below adenosine concentrations of 10−5 M. However, in the concentration range of 10−5 to 10−3 M, significant NAD reduction was obtained. Both calcium antagonists increased CVV markedly, but did not bring about significant changes in NAD/NADH ratio and local electrical activity of the exposed brain cortex. D-600 (2 × 10−6 M) increased CVV as much as did 10−4 M adenosine, but it failed to diminish the vascular and metabolic effects of the adenosine. D-600 (2 × 10−4 M) resulted in an increase in CVV approximately 2.5 times greater than that caused by 10−4 M adenosine alone. However, the adenosine-induced CVV response was inhibited by only about 70%, compared with the control response. After pretreating the brain cortex with 2 × 10−3 M D-600, adenosine had no effects on CVV and NAD/NADH redox state; the NAD reduction accompanying anoxia and epileptic seizures was considerably diminished. These results suggest that the inhibition of transmembrane calcium influx could have a minor role in the vasodilatatory mechanism of adenosine. Since the vascular effect of adenosine vanished only at very high concentration of D-600, which might also inhibit the release of calcium from intracellular binding sites, it is presumed that adenosine dilates the cerebrocortical vessels by interacting with intracellular calcium-sequestrating mechanisms. Furthermore, since adenosine had a marked NAD reducing effect and since it is well known that it increases the activity of adenylate cyclase and phosphorylase enzymes, accumulation of 3′,5′-cyclic adenosine monophosphate (cAMP) and substrate mobilization might be involved also in the vasodilatatory mechanism of adenosine. Our results concerning the inhibitory effect of D-600 on epilepsy- and anoxia-induced cerebrocortical NAD reduction unambiguously demonstrate the significance of calcium fluxes in glycogen and glucose metabolism under these conditions.