Inhibition of wild-type and mutant neuronal nicotinic acetylcholine receptors by local anesthetics.

Inhibition of neuronal nicotinic receptors can be regulated by the presence of specific amino acids in the beta subunit second transmembrane domain (TM2) domain. We show that the incorporation of a mutant beta4 subunit, which contains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, greatly reduces the sensitivity of receptors to the local anesthetic [2-(triethylamino)-N-(2,6-dimethylphenyl)acetamide] (QX-314). Although differing in potency, the inhibition of both wild-type alpha3beta4 receptors and alpha3beta4(6'F10'T) receptors by QX-314 is voltage-dependent and noncompetitive. Interestingly, the potency of the local anesthetic tetracaine for the inhibition of alpha3beta4 and alpha3beta4(6'F10'T) receptors seems unchanged when measured at -50 mV. However, whereas the onset of inhibition of wild-type alpha3beta4 receptors is voltage-dependent and noncompetitive, the onset of inhibition of alpha3beta4(6'F10'T) receptors by tetracaine is unaffected by membrane voltage, and at concentrations < or = 30 microM seems to be competitive with acetylcholine. This may be due to either direct effects of tetracaine at the acetylcholine binding site or preferential block of closed rather than open channels in the mutant receptors. Further analysis of receptors containing the 6' mutation alone suggests that although the 6' mutation is adequate to alter the voltage dependence of tetracaine inhibition, both point mutations are required to produce the apparent competitive effects.

A beta vasoactivity in vivo.

Bilateral temporoparietal hypoperfusion has been frequently observed early in the Alzheimer's disease (AD) process. The beta-amyloid (A beta) peptide is believed to play a central role in the pathogenesis of AD. In vitro experiments have shown that freshly solubilized A beta enhances constriction of cerebral and peripheral vessels. We proposed that in vivo, A beta would also have vasoactive properties. To test this hypothesis, we intraarterially infused freshly solubilized A beta 1-40 in rats and observed changes in peripheral blood pressure, cerebral blood flow, and cerebrovascular resistance. We found that infusion of A beta in vivo significantly increased the blood pressure in hypotensive rats but not in normotensive and hypertensive rats. Moreover, A beta infusion also resulted in a decreased blood flow and increased vascular resistance specifically in cerebral cortex but not in heart or kidneys. These data suggest that A beta has a direct and specific constrictive effect on cerebral vessels in vivo, which may contribute to the cerebral hypoperfusion observed early in the AD process.

Bcl-X(L) inhibits apoptosis and necrosis produced by Alzheimer's beta-amyloid1-40 peptide in PC12 cells.

Recent studies have shown that neuronal apoptosis induced by the Alzheimer's disease (AD) beta-amyloid peptide (Abeta) is related to alteration of the Bax/Bcl-2 ratio. It has been demonstrated that Bcl-X(L) (Bcl-X(L) = protein, bcl-X(L) = gene), a Bcl-2-related protein, prevents apoptosis in mammalian cells. Additionally, TGF-beta1 is able to protect cultured neuronal cells from Abeta-induced apoptosis via upregulation of bcl-X(L) and bcl-2 gene expression. We show that Abeta treatment (500 nM, freshly solubilized) results in apoptosis and necrosis in differentiated PC12 cells maintained with a low dose of NGF-beta (1 ng/ml). To investigate whether transfection of PC12 cells with bcl-X(L) could block Abeta-induced apoptosis, we transfected these cells with a bcl-X(L) construct (pcDNA-bcl-X(L)). Data show that bcl-X(L) significantly inhibits both early-stage apoptosis and late-stage apoptosis/necrosis produced by Abeta treatment (1000 nM) in pcDNA3-bcl-X(L)-transfected PC12 cells as compared with pcDNA3 vector-transfected PC12 cells. These results suggest that Bcl-X(L) exhibits both anti-necrotic as well as anti-apoptotic roles in Abeta-challenged PC12 cells.

Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis.

It is well known that microglial cells perform a key role in mediating inflammatory processes, which are associated with neurodegenerative diseases such as multiple sclerosis (MS). In this study, we report that CD40 expression on microglia is greatly enhanced by a low dose (10 U/ml) of IFN-gamma. We also find that ligation of microglial CD40 by CD40L triggers a significant production of TNF-alpha. Activation of microglia by ligation of CD40 in the presence of IFN-gamma results in cultured cortical neuronal injury, which is markedly attenuated by blockade of the CD40 pathway or neutralization of TNF-alpha. Finally, we find significant levels of IFN-gamma and TNF-alpha in the medium of co-cultured activated CD4+ T cells and microglial cells, showing that microglia can supply the CD40 receptor to activated CD4+ T cells and suggesting that this cellular interaction is a key event in MS pathophysiology.

Soluble Alzheimers beta-amyloid constricts the cerebral vasculature in vivo.

Bilateral temporoparietal hypoperfusion has been frequently observed early in the Alzheimer's disease (AD) process. An increased beta-amyloid (Abeta) peptide is believed to play a central role in the pathogenesis of AD. In vitro experiments have shown that freshly solubilized Abeta enhances constriction of cerebral and peripheral vessels. We propose that in vivo the Abeta vasoactive property may contribute to cerebral hypoperfusion of AD patients. To test this hypothesis, we intra-arterially infused freshly solubilized Abeta -40 in rats and observed changes in cerebral blood flow and cerebrovascular resistance using fluorescent microspheres. We found that infusion of Abeta in vivo resulted in a decreased blood flow and increased vascular resistance specifically in cerebral cortex but not in heart or kidneys. These data suggest that Abeta has a direct and specific constrictive effect on cerebral vessels in vivo, which may contribute to the cerebral hypoperfusion observed early in the AD process.

Alzheimer's beta-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40.

Accumulating evidence suggests that beta-amyloid (Abeta)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Abeta in the inflammatory processes in vascular tissues, we have tested the ability of Abeta to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Abeta dose-dependently increased the expression of CD40, and secretion of interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) in endothelial cells. Abeta also induced expression of IFN-gamma receptor (IFN-gammaR) both in endothelial and smooth muscle cells. Characterization of the Abeta-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-gammaR. Moreover, IL-1beta and IFN-gamma synergistically increased the Abeta-induced expression of CD40 and IFN-gammaR. We have recently found that Abeta induces expression of adhesion molecules, and that cytokine production and interaction of CD40-CD40 ligand (CD40L) further increase the Abeta-induced expression of adhesion molecules in these same cells. These results suggest that Abeta can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40-CD40L and cytokines. Additionally, Abeta1-42, the more pathologic form of Abeta, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Abeta1-40. Collectively, these findings support the hypothesis that the Abeta-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.