Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.

Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway.

While it has been shown that the angiotensin type-2 (AT(2)) receptor plays an important role in the development and differentiation of many tissues, the second messengers involved in its signaling pathways are just beginning to be understood. To further determine the signaling pathways for the AT(2) receptor, we have investigated whether human angiotensin type-2 receptor transfected into Chinese hamster ovary (CHO) cells can modulate insulin-induced extracellular signal-related protein kinase (ERK-2) phosphorylation via a G-protein coupled mechanism. Our results indicate that the human AT(2) receptor decreases insulin-induced ERK-2 phosphorylation through a G-protein mediated pathway since inhibition was attenuated by pertussis toxin (a G(i)/G(0) inhibitor). Our findings further indicate that the inhibitory response was insensitive to sodium orthovanadate (a PTPase inhibitor), but sensitive (attenuated) to okadaic acid, suggesting an important role for protein phosphatase 2A (PP2A). We have also shown that alanine substitution of the putative G-protein coupling DRY(141-143) motif of the second intracellular loop significantly decreases the human AT(2) receptor's ability to inhibit insulin-induced ERK-2 phosphorylation. Our results support the hypothesis that the AT(2) receptor inhibits insulin-induced ERK-2 activity via a G-protein coupled pathway involving the up-regulation of PP2A.

Effects of mutations in the highly conserved DRY motif on binding affinity, expression, and G-protein recruitment of the human angiotensin II type-2 receptor.

The signaling pathways for the seven transmembrane G-protein coupled angiotensin II receptors (AT(1) and AT(2)) are just beginning to be understood. While these receptors play an important role in the development and differentiation of many tissues, including the cardiovascular and central nervous systems, information about amino acid motifs involved in angiotensin II-mediated signaling is only available for the AT(1) receptor subtype. In the present study, we mutated the conserved DRY(141-143) motif in the AT(2) receptor, which is thought to be involved in G-protein recruitment. Expression of wild type and mutant receptors in CHO-K1 cell plasma membranes was confirmed using radioligand binding analyses. Our findings indicate a significant change in the binding affinities (kD) and capacities (B(max)) of the mutant receptors relative to wild type. Alanine substitutions of D(141) and DRY(141-143) resulted in a significant decrease of binding affinity for both Sar(1)Ile(8)-angiotensin II (SarIle-Ang II) (mixed agonist/antagonist) and angiotensin II (agonist). The binding affinities following alanine substitutions of R(142) and Y(143) were not significantly different from wild type receptor. Interestingly, the R(142)-A and Y(143)-A mutants revealed a significant decrease in binding levels from wild type with SarIle-Ang II, but not angiotensin II. The effect of GTPgammaS on angiotensin II binding affinity between wild type and mutant receptors was similarly significant. The D(141)-A, Y(143)-A, and DRY(141-143)-AAA mutant receptors showed a marked decrease in GTPgammaS-induced angiotensin II affinity shift. The R(142)-A GTPgammaS binding affinity shift was not different from the wild type receptor. Our results support the hypothesis that the DRY motif plays a significant role in the binding affinity, structural stability and G-protein recruiting of the AT(2) receptor.