Neuroprotective action of halogenated derivatives of L-phenylalanine.

BACKGROUND AND PURPOSE: The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. METHODS: We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). RESULTS: 3,5-diiodo-L-tyrosine (DIT) and 3,5-dibromo-L-tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC50s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6+/-14.1 micromol/L and 127.5+/-13.3 micromol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7+/-14.1% and 57.1+/-12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. CONCLUSIONS: DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Expression of angiotensin type 1 and 2 receptors in brain after transient middle cerebral artery occlusion in rats.

Angiotensin II (Ang II) type 2 receptors (AT2Rs) have been associated with apoptosis. We hypothesized that AT2Rs are increased in stroke and may contribute effects of stroke to the brain. To test this, we have examined the expression of Ang II type 1 receptor (AT1R), AT2R and Ang II levels in the brain 24 h after transient middle cerebral artery occlusion (MCAO). The densities of AT1R and AT2R were measured by quantitative autoradiography (n=6). The levels of Ang II were measured by radioimmunoassay (RIA) (n=6) and by immunohistochemistry (n=3). AT1R levels on autoradiography showed a significant decrease (0.87+/-0.06 to 1.39+/-0.07 fmol/mg, p<0.01) in the ventral cortex of the stroke side compared to the cortices of non-stroke (NS) rats (n=4). There was no significant difference on ATIR in the contralateral verbal cortex of the stroke rats compared to NS control. In contrast, levels of AT2R in the ventral cortex of both the stroke and the contralateral sides were significantly increased (0.77+/-0.06, p<0.05 and 0.91+/-0.05, p<0.01 compared to 0.60+/-0.03 fmol/mg tissue, respectively). RIA showed that Ang II in the ventral cortex of both the stroke and the contralateral sides were significantly increased (241.63+/-47.72, p<0.01 and 165.51+/-42.59, p<0.05 compared to 76.80+/-4.10 pg/g tissue, respectively). Also, Ang II in the hypothalamus was significantly increased (179.50+/-17.49 to 118.50+/-6.65 pg/g tissue, p<0.05). Immunohistochemistry confirmed the increase of Ang II. These results demonstrate that brain Ang II and AT2Rs are increased whereas AT1Rs are decreased after transient MCAO in rats. We conclude that in stroke, Ang II and AT2R are activated and may contribute neural effects to brain ischemia.

Antisense to epidermal growth factor receptor prevents the development of left ventricular hypertrophy.

We previously demonstrated that left ventricular hypertrophy (LVH) induced by angiotensin II infusion requires epidermal growth factor receptor (EGFR) activation to mediate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. To test whether the EGFR-mediated MAPK/ERK activation plays an important role in development and maintenance of LVH in spontaneously hypertensive rats (SHR), we investigated the effects of antisense oligodeoxynucleotide to EGFR (EGFR-AS) on LVH and blood pressure in young and adult SHR. EGFR-AS, sense oligonucleotide to EGFR (EGFR-S; 1.5 mg/kg), or vehicle control (5% dextrose) with liposome was injected once a week for 2 months in 5- or 13-week-old SHR. The effect of EGFR-AS on the expression of EGFR and phosphorylated ERK in the heart were examined by Western blots. After treatment, EGFR-AS significantly (P<0.05) decreased left ventricular weight/body weight and blood pressure in young SHR compared with EGFR-S or control-treated rats. In adult SHR, EGFR-AS did not affect left ventricular weight/body weight and blood pressure. EGFR and phosphorylated ERK significantly declined from 5 to 20 weeks (P<0.05). EGFR-AS, but not EGFR-S, significantly (P<0.05) decreased the expression of EGFR and phosphorylated ERK in young SHR, but had no significant effect in adult SHR. These results suggests that EGFR-mediated ERK activation is critically important for LVH in young SHR. This may be related to the high levels of EGFR and phosphorylated ERK in young SHR, suggesting a critical role of the EGFR-activated ERK pathway in cardiovascular development but not in the maintenance of established LVH in adult SHR.