Biomedical subjects

Tom N Grammatopoulos

Publications and source records attributed to Tom N Grammatopoulos.

8 recordsLinked to original sources

PubMed2007

Angiotensin type 1 receptor antagonist losartan, reduces MPTP-induced degeneration of dopaminergic neurons in substantia nigra.

BACKGROUND: Recent attention has focused on understanding the role of the brain-renin-angiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT1) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice (Fig. 1). RESULTS: In the presence of exogenous Ang II, losartan reduced MPP+ (5 muM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH+ immunostaining to 34% of control. CONCLUSION: Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT1 receptor as a potential novel target for neuroprotection.

Journal Article↗

PubMed2005

Angiotensin II protects cultured midbrain dopaminergic neurons against rotenone-induced cell death.

In this study, we demonstrate that angiotensin II (Ang II) protects dopamine (DA) neurons from rotenone toxicity in vitro. Primary ventral mesencephalic (VM) cultures from E15 rats were grown for 5 days and then cultured in the presence of the mitochondrial complex I inhibitor, rotenone. Acute exposure (20 h) to 20 nM rotenone reduced the number of tyrosine hydroxylase-positive (TH+) neurons by 50 +/- 6% when compared to untreated cultures. Pre-treatment of VM cultures with 100 nM Ang II decreased TH+ neuronal loss to 25 +/- 10% at the 20-nM rotenone concentration. Ang II in the presence of the angiotensin type 1 receptor (AT1R) antagonist, losartan, was even more effective in protecting DA neurons showing a loss of only 13 +/- 4% at 20 nM rotenone. Conversely, the AT2R antagonist, PD123319, abolished the protective effects of Ang II. Furthermore, both the NMDA receptor antagonist, MK801, and the antioxidant, alpha-tocopheryl succinate (vitamin E analogue), prevented rotenone-induced toxicity. Here, we show that acute exposure of VM cultures to the pesticide rotenone leads to dopaminergic neuronal cell death and that angiotensin acting through the AT2 receptor protects dopamine neurons from rotenone toxicity.

Angiotensin II↗

PubMed2004

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum.

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.

Alanine↗

PubMed2004

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.

Animals↗

PubMed2004

Angiotensin II attenuates chemical hypoxia-induced caspase-3 activation in primary cortical neuronal cultures.

In this study we determined whether caspase-3 is required in mouse cortical neurons for sodium azide-mediated apoptosis. Primary cortical neuronal cultures were treated with a cell permeable caspase-3 inhibitor, DEVD (1 nM-100 fM), prior to sodium azide-induced hypoxia. Treatment with the caspase-3 inhibitor resulted in a dose-dependent decrease in apoptosis, suggesting that sodium azide-induced apoptosis is mediated through a caspase-3 dependent pathway. Levels of cytochrome-c release and caspase-3 cleavage were assayed by Western analysis. Cytochrome-c release and caspase-3 cleavage were observed at 5 h (85.3+/-5.8%) and 8 h (53.4+/-14.9%), respectively. We have previously reported that angiotensin II, acting through the AT(2) receptor subtype, protects cultured mouse cortical neurons from sodium azide-induced apoptosis. We also examined whether the protective effect of angiotensin II is mediated through modulation of caspase-3. Pre-treatment of cells with angiotensin II and the AT(1) receptor antagonist, losartan, reduced levels of sodium azide-induced caspase-3 cleavage by 95.0+/-4.0%. Cells pre-treated with the AT(2) receptor antagonist, PD123319 showed a smaller reduction of caspase-3 cleavage (53.8+/-3.4%). Our findings indicate that sodium azide-induced apoptosis is caspase-3 dependent and that angiotensin II protects cortical neurons from chemical-induced apoptosis by reducing caspase-3 cleavage.

Angiotensin II↗

PubMed2004

Angiotensin type 2 receptor neuroprotection against chemical hypoxia is dependent on the delayed rectifier K+ channel, Na+/Ca2+ exchanger and Na+/K+ ATPase in primary cortical cultures.

We have previously reported that angiotensin II (Ang II) protects cortical neurons from chemical-induced hypoxia through activation of the angiotensin type 2 (AT(2)) receptor. Here, we show in mouse primary neuronal cultures that the AT(2) receptor neuroprotection results from the activation of the delayed rectifier K(+) channel as well as the involvement of the Na(+)/Ca(2+) exchanger (NCX) and Na(+)/K(+) ATPase (ATPase). Roles of the K(+) channel, NCX and ATPase were determined using the specific blockers alpha-dendrotoxin, KB-R7943 and ouabain, respectively. Sodium azide (10mM) induced apoptosis in 40% of neurons. Inhibition of the AT(1) receptor with losartan (1 microM) facilitated angiotensin II mediated neuroprotection by reducing sodium azide-induced apoptosis 61.8 +/- 5.6%, while inhibition of the AT(2) receptor with PD123319 (1 microM) showed no neuroprotection. These results suggest that angiotensin II neuroprotection is mediated through the AT(2) receptor and requires inhibition of the AT(1) receptor in order to facilitate its effect. To determine the roles of delayed rectifier K(+) channel, NCX and ATPase cultures were pretreated with alpha-dendrotoxin (10nM), KB-R7943 (100 nM) and ouabain (100 nM), which significantly attenuated AT(2) receptor mediated neuroprotection. These findings further suggest that the mechanism of AT(2) receptor mediated neuroprotection is coupled to activation of the delayed rectifier K(+) channel, NCX and ATPase.

Animals↗

PubMed2003

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons.

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.

Animals↗

PubMed2002

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.

Amino Acid Motifs↗