Dong-Mei Zuo

Oxidative Deamination of Methylamine by Semicarbazide-Sensitive Amine Oxidase Leads to Cytotoxic Damage in Endothelial Cells: Possible Consequences for Diabetes

Methylamine was observed to be deaminated by several semicarbazide-sensitive amine oxidases, which were prepared from blood and vascular tissues of various species, including humans. Although methylamine itself is relatively nontoxic toward endothelial cells obtained from both human umbilical vein and calf pulmonary artery, it becomes very toxic in the presence of SSAO. SSAO inhibitors (i.e., MDL-72974A) effectively protected the cells from methylamine-SSAO–induced damage. The cytotoxicity seems, therefore, to be a consequence of the deamination of methylamine. Our findings suggest that formaldehyde, the deaminated product of methylamine, may be responsible for these toxic effects. Human serum, which also contains SSAO, was also capable of deaminating methylamine and causing cytotoxicity to cultured endothelial cells. Both methylamine and SSAO circulate in human blood, and their concentrations in the blood of normal healthy subjects are quite close to those required to induce cytotoxicity in tissue-cultured cells. Both SSAO activity and methylamine levels have been reported to be increased in the blood of diabetic individuals. Blood SSAO activity also has been reported to be elevated in the blood of STZ-induced diabetic rats. It is possible, therefore, that an abnormal metabolism of methylamine may be involved in endothelial injury, and that it may subsequently induce atherosclerotic plaque formation and thus be involved in the cardiovascular disorders seen in diabetes.

Formation of Formaldehyde from Adrenaline In Vivo; a Potential Risk Factor for Stress-Related Angiopathy

Cardiovascular and cerebrovascular disorders are well known to be associated with stress related behaviors. Stress enhances excretion of adrenaline, which is deaminated by monoamine oxidase and methylamine is formed. This product can be further deaminated by semicarbazide-sensitive amine oxidase (SSAO) and converted to toxic formaldehyde, hydrogen peroxide and ammonia. SSAO is located in the cardiovascular smooth muscles and circulated in the blood. We investigated whether formaldehyde can be derived from adrenaline in vivo. Methylamine was confirmed to be a product of adrenaline catalyzed by type A monoamine oxidase (MAO-A). Irreversible and long-lasting radioactive residual activity was detected in different tissues following administration of 1-[N-methyl-3H]-adrenaline. Such irreversible linkage could be blocked by selective MAO-A or SSAO inhibitors. Endothelial cells are quite sensitive to formaldehyde and relatively resistant to hydrogen peroxide. It is possible that stimulation of adrenaline excretion by chronic stress could increase the levels of circulatory formaldehyde. Such chronic “formaldehyde” stress may be involved in the initiation of endothelial injury and subsequently angiopathy.

Differential effects of staurosporine and retinoic acid on the vulnerability of the SH‐SY5Y neuroblastoma cells: Involvement of Bcl‐2 and p53 proteins

Human catecholaminergic neuroblastoma cells (SH‐SY5Y) have been widely used in different neurochemical investigations. Quite often these cells are induced to differentiation by various agents, such as staurosporine and retinoic acid. Interestingly, even though both staurosporine and retinoic acid induce similar morphological differentiation in SH‐SY5Y cells, we found that these two groups of differentiated cells exhibited opposite vulnerability to harmful chemicals and physical insults. In the present study, cisplatin, 5‐fluorouracil (5‐FU), N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4), 6‐hydroxydopamine (6‐OHDA), and γ‐radiation were used to assess the tolerance of the differentiated cells. Cell viability was determined by 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Staurosporine‐treated SH‐SY5Y cells were more sensitive to these toxic insults than the untreated controls. In contrast, retinoic acid‐treated cells became more resistant to the same treatments. The expression of the proteins of the protooncogene Bcl‐2 and the tumor suppressor gene p53 following staurosporine or retinoic acid treatment was assessed by Western blot and immunocytochemistry. Retinoic acid increased Bcl‐2 and decreased p53 levels, whereas staurosporine decreased Bcl‐2 and increased p53 levels. The opposite alteration of Bcl‐2 (anti‐apoptotic) and p53 (apoptotic) contents in SH‐SY5Y cells with retinoic acid and staurosporine are attributed to the changes in cell vulnerability. These observations also indicate that caution should be taken when chemically induced differentiated neuroblastoma cells are to be used as an in vitro model for studying neuronal survival. J. Neurosci. Res. 58:426–435, 1999.

Characterization of human serum and umbilical artery semicarbazide-sensitive amine oxidase (SSAO): Species heterogeneity and stereoisomeric specificity

Semicarbazide-sensitive amine oxidases (SSAOs) are located in cardiovascular smooth muscle, cartilage and brown adipose tissues of different species, including human. The enzyme is also present in blood, and its activity appears to be altered under certain pathological conditions. SSAOs from both human umbilical arteries and serum were partially purified, and some of their biochemical properties were investigated. Both human artery and blood SSAO exhibited very similar substrate preference, lack of stereospecificity catalyzing the deamination of pro-R and pro-S benzylamine-deuterated enantiomers, and were very sensitive towards (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A). It was concluded that circulating serum SSAO is identical to the SSAO from vascular tissues. Human SSAO exhibited distinctly different properties in comparison to bovine and rat SSAOs.

Semicarbazide-sensitive amine oxidase and monoamine oxidase in rat brain microvessels, meninges, retina and eye sciera

Monoamine oxidase-A and -B (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) activities were assessed in several rat micro-vascular tissues and eyes using selective substrates and inhibitors. In rat brain microvessels both MAO-A and MAO-B activities are relatively high and the levels of the two types of MAOs are comparable. Retina possesses a similar ratio of MAO-A and B but the activities are much lower. Eye sclera and meninges exhibit mainly MAO-A and MAO-B, respectively. Aorta is the only tissue where SSAO is the predominant amine oxidase. Relatively low, but significant amounts of SSAO were also detected in brain microvessels, meninges, retina and eye sclera. Methylamine was observed to be deaminated by SSAO from different tissues. The physiological and toxicological implications of amine oxidases in these tissues are discussed.

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The IC50 values were estimated to be 2 x 10(-9) M, 5 x 10(-9) M, 8 x 10(-8) M and 2 x 10(-8) M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A (IC50 = 3 x 10(-7) M. Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ED50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.

Immunohistochemical evidence of neuroprotection by R(−)‐deprenyl and N‐(2‐hexyl)‐N‐methylpropargylamine on DSP‐4‐induced degeneration of rat brain noradrenergic axons and terminals

DSP‐4 [N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzyl‐amine] is a potent neurotoxin highly selective to the locus coeruleus noradrenaline (NA) system. Previous biochemical studies have shown that the monoamine oxidase‐B (MAO‐B) inhibitors, R(−)‐deprenyl and (±)2‐HxMP [N‐(2‐hexyl)‐N‐methylpropargylamine], are able to prevent DSP‐4 induced NA depletion in the mouse hippocampus. It is not quite certain, however, whether this actually represents neuroprotection of NA axons or a metabolic effect due to inhibition of MAO activity. Employing dopamine‐β‐hydroxylase immunohistochemical and image analysis methods, we have shown that 92% and 84% of NA nerve fibers in the rat hippocampus are spared from DSP‐4 neurotoxicity by a single pretreatment dose of either R(−)‐deprenyl or (±)2‐HxMP respectively. Similar neuroprotective effects of R(−)‐deprenyl and (±)2‐HxMP were also observed in the cerebral cortex, thalamus, amygdaloid complex and cerebellum. This is the first morphological evidence demonstrating that R(−)‐deprenyl and (±)2‐HxMP can indeed protect noradrenergic axons of locus coeruleus origin against DSP‐4 neurotoxicity.

Neuroprotection by R(−)-deprenyl and N-2-hexyl-N-methylpropargylamine on DSP-4, a neurotoxin, induced degeneration of noradrenergic neurons in the rat locus coeruleus

N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a neurotoxin and capable of selectively depleting noradrenergic axons and subsequently causing lesions of locus coeruleus (LC) noradrenergic neurons in the rat. R(-)-deprenyl and N-(2-hexyl)-N-methylpropargylamine (2-HxMP) have been previously shown to be quite effective in protecting NA nerve fibers in different brain tissues against DSP-4. The present report reveals the neuroprotective effect of these drugs on the LC noradrenergic cell bodies using a histochemical method. Neurons were quantitatively assessed using Nissl-stained sections. DSP-4 induced a 34% loss of LC perikarya neurons 3 months after a single systemic administration in comparison to control animals. Approximately 90% and 88% of neurons in the same regions survived against DSP-4 induced insult following multiple injections of R(-)-deprenyl and 2-HxMP, respectively. The neuroprotective effect towards the LC neurons against DSP-4 is probably due to prevention of retrograde degeneration of NA axons.

MK-801 INDUCES APOPTOTIC NEURONAL DEATH IN THE RAT RETROSPLENIAL CORTEX : PREVENTION BY CYCLOHEXIMIDE AND R(-)-2-HEXYL-N-METHYLPROPARGYLAMINE

MK‐801 is a non‐competitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist which can prevent excitatory neuronal death. At higher concentrations, however, it can also induce neuronal death in the limbic system. This MK‐801‐induced selective neurotoxicity has been proposed as an animal model for dementia and psychosis. We have investigated the effects of the protein synthesis inhibitor cycloheximide and the neurorescue agent 2‐hexyl‐N‐methylpropargylamine [R(−)‐2HxMP] on MK‐801‐induced neuronal death in the retrosplenial cortex in the rat. Cycloheximide [2 mg/kg, subcutaneously (sc)] administered either 1 hr before, or after, injection of MK‐801 (5 mg/kg, sc) prevented almost completely neuronal shrinkage and nuclear condensation of the granular retrosplenial cortex as assessed by hematoxylin‐eosin staining. The results suggest that the MK‐801‐induced neuronal death was apoptotic. This neurorescue effect by cycloheximide was time dependent: after 4 hr the effect was reduced to about 50% and by 8 hr had disappeared. R(−)‐2HxMP (0.25 mg/kg, sc), which does not inhibit protein synthesis in vitro, was also found to be effective at preventing MK‐801‐induced neuronal death.

Neurochemical, neuroprotective and neurorescue effects of aliphatic N-methylpropargylamines; new MAO-B inhibitors without amphetamine-like properties.

A series of aliphatic N-methylpropargylamine MAO-B inhibitors have been synthesized and their structural and functional relationships have been investigated. 2-Hexyl-N-methylpropargylamine (2-HxMP), for example, has been found to be a highly potent, irreversible, selective, MAO-B inhibitor both in vitro and in vivo. The R-(-)-enantiomers are much more active than the S-(+)-enantiomers at inhibiting MAO-B activity. Some of these compounds protect mouse nigrostriatal dopamine neurons against the neurotoxin MPTP and the mouse hippocampal noradrenergic system against the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). They rescue hippocampal neurons after damage induced by ischemia and kainic acid treatment, as well as motoneurons in young mice following facial nerve axotomy. Such rescue effects are, interestingly, unrelated to inhibition of MAO-B activity. Some of the aliphatic propargylamines enhance the survival of neuroblastoma cells co-cultured with astrocytes following serum depletion. They stimulate the expression of AADC mRNA and inhibit GFAP mRNA expression. They do not possess amphetamine-like properties and exhibit no effect on noradrenaline or dopamine uptake nor do they increase hypertensive effects in the tyramine pressor test. Unlike R(-)-deprenyl, 2-HxMP does not potentiate dopamine toxicity in vitro. These new MAO-B inhibitors may possess significant chemotherapeutic implications for certain psychiatric and neurodegenerative disorders.