Robert M. Hysmith

A role for a new vascular enzyme in the metabolism of xenobiotic amines.

Although it has long been thought that environmental toxins may play an underlying role in vascular diseases such as atherosclerosis, this concept is not supported by any clear-cut experimental evidence of toxic metabolism by cardiovascular enzymes. In this study, we demonstrate that allylamine, a selective cardiovascular toxin in vivo, is actively metabolized in vitro by a purified vascular enzyme (semicarbazide-sensitive amine oxidase), which has been localized recently to vascular smooth muscle cells. Oxidative deamination of allylamine to a highly toxic aldehyde, acrolein, was blocked through enzyme inhibition by semicarbazide-sensitive amine oxidase suggests that this vascular enzymes physiological role may include metabolism of exogenous amines.

In vitro expression of benzylamine oxidase activity in cultured porcine smooth muscle cells.

Benzylamine oxidase (BzAO) activity was measured in cultured smooth muscle and endothelial cells derived from thoracic aorta of adult swine, and in homogenates of dissected aortic medial and intimal layers. BzAO appeared to be localized in aortic medial homogenates. Cultured smooth muscle cells (which were verified by electron microscopic characteristics and positive staining with antimyosin) showed high BzAO activity, with optimal activity at pH 7.0; no activity was detected in endothelial cells. Prolonged serial passage of smooth muscle cells resulted in increased BzAO activity, with the increase most marked by the 12th population doubling. Also, BzAO was found predominantly in the soluble portion of the culture medium of smooth muscle cells by their 12th population doubling. These findings support the concept that vascular BzAO is localized within medial smooth muscle cells, and suggest that smooth muscle cells may be capable of secreting BzAO.

Role of benzylamine oxidase in the cytotoxicity of allylamine toward aortic smooth muscle cells

In this study we demonstrate that by inhibiting benzylamine oxidase (BzAO) with either semicarbazide or phenelzine, aortic smooth muscle cells (ASMCs) are protected from cytolethal injury by the cardiovascular toxin allylamine. We find that although both semicarbazide and phenelzine inhibit BzAO or ASMCs grown in vitro, phenelzine is the more effective inhibitor. We further demonstrate that although semicarbazide--at concentrations inhibiting BzAO--protects ASMCs from cytolethal concentrations of allylamine, it does not fully protect ASMCs from sublethal injury as assessed by [3H]uridine uptake. In contrast, phenelzine appears to afford complete protection of ASMCs from allylamine injury. Although semicarbazide and phenelzine pretreatment does not interfere with [14C]allylamine uptake by ASMCs, retention time of the 14C-moiety from radiolabeled allylamine is less in pretreated ASMCs. Subcellular distribution studies of ASMCs exposed to [14C]allylamine demonstrate that inhibiting BzAO activity in ASMCs results in marked derangement of the distribution pattern of 14C-moiety in subcellular fractions of ASMCs, with 14C-moiety not localized to mitochondrial/endoplasmic reticulum enriched fractions.

Comparative toxicity of the cardiovacular toxin allylamine to porcine aortic smooth muscle and endothelial cells

This study supports a recent hypothesis that the cardiovascular toxin, allylamine, is toxic to smooth muscle cells of large elastic arteries (aorta). Cultures of the porcine aortic smooth muscle, endothelial, and fibroblastic cells were exposed to varying concentrations of allylamine ranging from 5 μM to 340 μM. Monitored cytotoxic and cytolytic activities demonstrated that final concentrations of 60 μM allylamine decreased cell population viability of smooth muscle cells as much as 50%. Viability decreased approximately linearly with increasing concentrations of allylamine including spontaneous lysing of smooth muscle cells at 90 μM. Endothelial cells were more resistant to lower concentrations of allylamine requiring 90 μM to decrease cell population viability by 50%. In contrast, fibroblastic cells were very resistant to lower concentrations of allylamine. The specific lytic response of these cells in culture, measured by release of [3H]thymidine, gave findings parallel to the viability studies, i.e. at 100 μM allylamine smooth muscle cells demonstrated 75% specific lysis while endothelial cells showed 29%. Growth studies of cells surviving an 8-h exposure to allylamine indicate that surviving endothelial cells have better growth characteristics than surviving smooth muscle cells; both cell lines are also apparently injured at concentrations of allylamine much lower than the CT50. These studies show that of the cellular components of the vascular wall, smooth muscle cells appear to be the most sensitive to the toxic effects of allylamine.

Allylamine cardiovascular toxicity: VI. subcellular distribution in rat aortas

The cardiovascular toxin allylamine (3-aminopropene) has been shown to concentrate in elastic and muscular tissues. In this study the 14C-moiety of [14C]allylamine was traced in aortas of adult Sprague-Dawley rats after intravenously injecting 30 microCi of [14C]allylamine (spec. act. = 0.4 mCi/mM). At 5, 10, 15 and 20 min after injection 33.3-29.8% of the 14C-moiety was sequestered in aortas; at 30 min 16.8% was still present. Subcellular fractionation of the postnuclear supernatant by isopycinic centrifugation in sucrose demonstrated that 5 min after administration of [14C]allylamine, the 14C-moiety displayed a modal density peak of 1.20 g/ml. Similar activities were observed up to 30 min exposure. This modal density was similar to the distribution pattern of mitochondria based on analysis of malate dehydrogenase activities. As early as 20 min post-exposure, mitochondrial malate dehydrogenase activities of aortic mitochondria decreased, while cytosolic malate dehydrogenase activities increased, suggesting mitochondrial membrane perturbation. We suggest that the subcellular site for allylamine injury to the aorta is the mitochondrion.

Binding of [14C] allylamine to isolated mitochondria from rat heart and aorta

This study demonstrates specific and saturable binding of [14C] allylamine to mitochondria derived from rat aorta and heart. Specific binding is linear with respect to mitochondrial concentration and has a pH optimum of 7.0. Saturation isotherms reveal anomalous kinetics of specific binding on heart mitochondria with a high affinity site (KD 16 nM) and a lower affinity site (KD 80 nM); Scatchard plots have a common intercept. Exhaustive flow dialysis in the presence of SDS demonstrates that as much as 23.5% of bound radioactive moieties in aorta mitochondria are covalently bound, and as much as 42.6% are covalently bound in heart mitochondria. Hydrolysis of heart mitochondria with phospholipase C markedly enhances saturation of [14C] allylamine, and greatly increases the quantity of covalently bound radioactive ligand. Phospholipase C hydrolysis of heart mitochondria increased monoamine oxidase B activities and unmasked a small amount of benzylamine oxidase activity, whereas hydrolysis of mitochondria with phospholipases A2 and D diminish MAO-B activity. The monoamine oxidase B inhibitor, deprenyl, significantly reduced both specific and covalent binding of the 14C-activity from [14C] allylamine to phospholipase hydrolyzed mitochondria. The benzylamine oxidase inhibitor, phenelzine, significantly decreased specific binding but had no effect on the degree of covalent binding of [14C] allylamine to phospholipase C hydrolyzed mitochondria. The benzylamine oxidase inhibitor, semicarbazide, had no effect in inhibiting [14C] allylamine binding. Covalent binding of 14C-moiety from [14C] allylamine to mitochondria--which express specific binding sites for the [14C] allylamine--and inhibition of binding by monoamine oxidase inhibitors, suggest the formation of highly reactive intermediates.