Differentiation between calcium antagonists in vitro and their effects in models of cerebral ischaemia.
thesisposted on 19.11.2015, 08:43 by Barry. Kenny
Different classes of calcium antagonist were defined in vitro using radioligand binding studies and the efficacy of the classes as neuroprotective agents was assessed in vivo using a novel model of cerebral ischaemia. Radioligand binding studies indicated that the interactions of both class I (dihydropyridines) and class II (verapamil and diltiazem) calcium antagonists was temperature-, ligand- and tissue-dependent. Specific binding sites for class III antagonists (flunarizine, fluspirilene, etc.), labelled by [3H] fluspirilene in skeletal muscle, were not identified in brain membranes, although these compounds allosterically regulated [3H] dihydropyridine binding in brain. All class III compounds displayed high affinity for the [3H] fluspirilene site in skeletal muscle membranes and thus fluspirilene appeared to be prototypical of this class of compound. The calcium antagonist SR 33557 identified a novel high affinity binding site in brain membranes. This putatative fourth site for a calcium antagonist appeared to be tightly coupled to the dihydropyridine site. Class III calcium antagonists displayed a range of affinities for other receptor and ion channel sites but all class III calcium antagonists showed high affinity for rat brain sodium channels labelled by [3H] batrachotoxinin-A-20-a-benzoate. In the Mongolian gerbil, forebrain ischaemia (10 min but not 5 min) with 7 days recovery produced a significant reduction in the number of [3H] PN 200-110 binding sites in hippocampal membranes. However, an w3 ligand, [3H] PK 11195 was shown to be a better marker of ischaemic damage. Binding experiments and autoradiographic analysis demonstrated a significant ischaemia-induced increase in the density of w3 sites after 5 min forebrain ischaemia and 7 days recovery in the gerbil. The distribution and pharmacology of w3 sites was very species dependent. [3H] PK 11195 was successfully used to establish a model of focal ischaemia in the mouse, in which a variety of class III calcium antagonists were found to be neuroprotective. The anticonvulsant phenytoin was active in the mouse focal ischaemia model and in an in vitro model of ischaemia (using the rat hippocampal slice). The interaction of calcium antagonists with both Na+ and Ca2+ channels is proposed to be important as a mechanism whereby these agents confer neuroprotection.