kn-93 and Stroke

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca(2+)/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca(2+)-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Cell Death; Cerebral Cortex; Gene Expression Regulation, Enzymologic; Gene Products, tat; Hippocampus; Mice; Molecular Sequence Data; Neurons; Neuroprotective Agents; Peptide Fragments; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Stroke; Sulfonamides

We studied the early pathophysiological response of lenticulostriate arterioles in rats in three models of human conditions associated with stroke: (a) chronic angiotensin II-hypertension; (b) chronic nicotine administration; (c) oxidative endothelial injury. In all three models, quantitative patch clamp analysis of freshly isolated vascular smooth muscle cells from lenticulostriate arterioles and posterior cerebral arteries showed significant increases in activity of functional L-type calcium channels that were due to an increase in open channel probability, with no change in other biophysical properties or in channel expression. In addition, all three models showed evidence of endothelial dysfunction, but of a different nature in the three. With chronic angiotensin II-hypertension, but not in the other two models, endothelial nitric oxide synthase (eNOS) was dysfunctional, was mislocalized away from its normal abluminal location, and was accumulated in peri-nuclear Golgi. By contrast, the other two models showed no mislocalization of eNOS, but instead showed evidence of oxidative stress in endothelium, with up-regulation of superoxide dismutase and hexose kinase. All three models showed significant up-regulation of expression of proliferative cell nuclear antigen (PCNA) (PCNA index, 70-80%) in arterioles in situ, which is associated with increased activation of the nuclear transcription factor, phospho-cAMP response element binding protein (phospho-CREB). In addition, calmodulin-dependent protein (CaM) kinase II was activated, in concert with the activation of L-type calcium channels. Furthermore, blockers of either L-type calcium channels (amlodipine) or of CaM kinase II (KN-93) completely prevented the activation of CREB and the up-regulation of PCNA in arterioles. Our findings demonstrate that abnormal regulation of L-type calcium channels is directly responsible for abnormal proliferative responses in vascular smooth muscle in various forms of cerebral arteriolar injury associated with endothelial dysfunction.

Topics: Amlodipine; Angiotensin II; Animals; Arterioles; Benzylamines; Brain; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; CREB-Binding Protein; Disease Susceptibility; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Hyperplasia; Hypertension; Models, Animal; Muscle, Smooth, Vascular; Nicotine; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nuclear Proteins; Oxidative Stress; Proliferating Cell Nuclear Antigen; Rats; Stroke; Sulfonamides; Trans-Activators