Monoamine oxidase and semicarbazide-sensitive amine oxidase kinetic analysis in mesenteric arteries of patients with type 2 diabetes

Monoamine oxidase (MAO, type A and B) and semicarbazide-sensitive amine oxidase (SSAO) metabolize biogenic amines, however, the impact of these enzymes in arteries from patients with type 2 diabetes remains poorly understood. We investigated the kinetic parameters of the enzymes to establish putative correlations with noradrenaline (NA) content and patient age in human mesenteric arteries from type 2 diabetic patients. The kinetic parameters were evaluated by radiochemical assay and NA content by high-performance liquid chromatography (HPLC). The activity of MAO-A and SSAO in type 2 diabetic vascular tissues was significantly lower compared to the activity obtained in non-diabetic tissues. In the correlation between MAO-A (K(m)) and NA content, we found a positive correlation for both the diabetic and non-diabetic group, but no correlation was established for patient age. In both groups, MAO-B (V(max)) showed a negative correlation with age. The results show that MAO-A and SSAO activities and NA content of type 2 diabetic tissues are lower compared to the non-diabetic tissues, while MAO-B activity remained unchanged. These remarks suggest that MAO-A and SSAO may play an important role in vascular tissue as well as in the vascular pathophysiology of type 2 diabetes.     

Cold-restraint induced gastric lesions in normotensive and spontaneously hypertensive rats

Spontaneously hypertensive (SHR) rats and normotensive Wistar-Kyoto (WKY) rats were subjected to 2 hr of cold-restraint stress at 2–6°C following a 24 hr fast. WKY rats had a significantly greater incidence and degree of ulceration of the gastric glandular mucosa than did SHR rats. Mean arterial pressure, obtained from a chronic arterial cannula, fell during 2 hr of cold-restraint stress in both SHR and WKY rats. Heart rate was unchanged in WKY but fell significantly in SHR. Plasma norepinephrine (NE) and epinephrine (E), determined by radioenzymatic assay, increased significantly following stress. Increased levels of NE remained similar for both SHR and WKY rats, while post-stress levels of E for the SHR rats greatly exceeded E levels for WKY rats. A greater degree of hypothermia was also noted in SHR rats. Decreased stress induced ulcerogenesis in the SHR may be due to the well-known altered hemodynamic and autonomic nervous system reactivity in this strain or other factors not yet discovered.     

Comparison of somatosympathetic reflex in normotensive and spontaneously hypertensive rats

In chloralose anaesthetized and paralyzed normotensive (Wistar, Wistar--Kyoto) and spontaneously hypertensive rats (SHR), a somatosympathetic reflex in the cervical sympathetic trunk elicited by a single electrical shock to forelimb afferent fibres in the median nerve was recorded. It has been shown that the elicited response of SHR is similar to the response of normotensive rats. Amplitude of somatosympathetic reflex in SHR is larger than that of somatosympathetic reflex in normotensive animals. It is supposed that somatosympathetic reflex in hypertensive and normotensive rats is formed in the same way. However, reflex excitability of sympathetic nervous system in SHR is greater.     

Culture medium enhances semicarbazide-sensitive amine oxidase activity

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.     

Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats

Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers.     

Physiological and pathological implications of semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of primary amines. Such deamination has been shown capable of regulating glucose transport in adipose cells. It has been independently discovered that the primary structure of vascular adhesion protein-1 (VAP-1) is identical to SSAO. VAP-1 regulates leukocyte migration and is related to inflammation. Increased serum SSAO activities have been found in patients with diabetic mellitus, vascular disorders and Alzheimer's disease. The SSAO-catalyzed deamination of endogenous substrates, that is, methylamine and aminoacetone, led to production of toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia, respectively. These highly reactive aldehydes have been shown to initiate protein cross-linkage, exacerbate advanced glycation of proteins and cause endothelial injury. Hydrogen peroxide contributes to oxidative stress. 14C-methylamine is converted to 14C-formaldehyde, which then forms labeled long-lasting protein adduct in rodents. Chronic methylamine treatment increased the excretion of malondialdehyde and microalbuminuria, and enhanced the formation of fatty streaks in C57BL/6 mice fed with an atherogenic diet. Treatment with selective SSAO inhibitor reduces atherogenesis in KKAy diabetic mice fed with high-cholesterol diet. Aminoguanidine, which blocks advanced glycation and reduces nephropathy in animals, is in fact more potent at inhibiting SSAO than its effect on glycation. It suggests that SSAO is involved in vascular disorders under certain pathological conditions. Although SSAO has been known for several decades, its physiological and pathological implications are just beginning to be recognized.     

Pressor responses to endothelin-1 in normotensive and spontaneously hypertensive rats

In freely moving rats, endothelin-1 (0.0135–4.5 nmol/kg) administered as an intravenous bolus injection, produced an immediate, short-lasting, dose-related fall in blood pressure followed by a long-lasting, dose-related increase in blood pressure. There was a higher sensitivity in the pressor responses to endothelin-1, in spontaneously hypertensive (SH) rats (ED₅₀ = 0.11 ± 0.02 and 0.28 ± 0.02 nmol/kg, in SH and normotensive rats, respectively), but no change in the maximal pressor effect of endothelin-1 in SH rats.

In rat isolated aorta, endothelin-1 induced a greater vasocontractile effect in SH rats than in normotensive rats. In both rat strains, removal of the endothelium did not change the concentration-effect curves obtained in endothelium-intact preparations. These data add further support to the hypothesis that endothelin-1 could play a role in genetic hypertension, at least in the maintenance of high blood pressure.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

The inhibition of semicarbazide-sensitive amine oxidase by aminohexoses

Semicarbazide-sensitive amine oxidase (EC 1.4.3.6; amine:oxygen oxidoreductase (deaminating) (copper-containing); SSAO) is a multifunctional protein. It acts under inflammatory conditions as a vascular-adhesion protein (VAP-1), mediating the adhesion of lymphocytes to vascular endothelial cells. The relationships, if any, between this adhesion function and the enzymatic functions (amine-substrate specificity and catalysis) of SSAO have not yet been defined. Since cell surface amino sugars and their derivatives are known to be involved in cell-to-cell recognition, we have investigated their possible effects on the enzyme activity of SSAO. The aminohexoses galactosamine, glucosamine and mannosamine were not oxidatively deaminated by SSAO. However, their presence during the assay of benzylamine oxidation resulted in a time-dependent inhibition. This inhibition was shown to follow saturation kinetics with respect to hexosamine concentration. Although time-dependent, the inhibition of SSAO activity was found to be reversible by dilution. In contrast, there is no such inhibition when the N-acetylamino sugar derivatives or the parent sugars (galactose, glucose and mannose) replaced the amino sugars in the reaction mixture. These results suggest that the interactions between SSAO and aminohexoses are specific and, therefore, that the cell-adhesion functions and amine-recognition functions of VAP-1/SSAO may be interlinked.     

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.     

Stimulation of glucose transport by semicarbazide-sensitive amine oxidase activity in adipocytes from diabetic rats

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO such as benzylamine in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in mouse 3T3-L1 adipocytes and in isolated rat adipocytes. Here we examined whether this combination of molecules also stimulates glucose transport in adipocytes from streptozotocin-induced diabetic rats and from Goto-Kakizaki diabetic rats. As previously reported, adipocytes obtained from streptozotocin-induced diabetic rats, showed a reduced stimulation of glucose transport in response to insulin. Under these conditions, the combination of benzylamine and vanadate caused a marked stimulation of glucose transport that was similar to the stimulation detected in control adipocytes. Adipocytes isolated from Goto-Kakizaki diabetic rats also showed a defective response to insulin; however, acute incubation in the presence of benzylamine and vanadate stimulated glucose transport in these cells to the same extent than in adipocytes from non-diabetic rats. These data indicate that adipocytes obtained from two different models of animal diabetes do not show resistance to the activation of glucose transport by SSAO activity, which is in contrast to the well reported resistance to insulin action. It seems to suggest that SSAO activity in combination with vanadate triggers a glucose transport-activating intracellular pathway that remains intact in the diabetic state. Further, our data support the view that the combination of benzylamine and vanadate could be an effective therapy in diabetes.     

Central monoamine oxidase and phenolsulfotransferase activities in spontaneously hypertensive rats

The activities of monoamine oxidase and phenolsulfotransferase in the hypothalamus and anterior pituitary gland of spontaneously hypertensive rats and the normotensive control (Wistar Kyoto rat) rats were investigated. The monoamine oxidase activity (determined using dopamine as substrate) in both these tissues was not significantly different between the normo- and hypertensive animals. Hypothalamic phenolsulfotransferase does not sulfate-conjugate dopamine at pH of 6.5 and pituitary phenolsulfotransferase does not sulfate-conjugate dopamine or 3,4-dihydroxyphenylacetic acid at the same pH. Hypothalamic phenolsulfotransferase activity determined using 3,4-dihydroxyphenylacetic acid as substrate was significantly higher in the spontaneously hypertensive than the Wistar Kyoto rats, while pituitary enzyme (determined using phenol as substrate) was the same in both strains of animals. We proposed that in the spontaneously hypertensive rats the higher level of hypothalamic phenolsulfotransferase could (by removing 3,4-dihydroxyphenylacetic acid as sulfated acid) increase the deamination of dopamine by monoamine oxidase. This could in turn result in the presence of high amount of sulfated 3,4-dihydroxyphenylacetic acid in the anterior pituitary gland reported in our earlier study, and be partly responsible for the reduced central dopaminergic activity found in the hypertensive rats.     

Tissue distribution of acebutolol in mature normotensive and stroke-prone spontaneously hypertensive rats

Tissue distribution of acebutolol was studied in 33-week-old normotensive (WKY) and Okamoto stroke-prone (SHR-SP) rats, 30 min after an i.v. administration, by using 14C-acebutolol. Plasma level of acebutolol was higher in WKY than in SHR-SP. Aorta, kidney, liver and muscle radioactivity/plasma radioactivity ratios were higher in SHR-SP than in WKY. The brain/plasma radioactivity ratio was very low and similar in the two groups. The drug distribution was the same in the two groups except in medulla + corpus trapezoides where drug concentration was greater in SHR-SP. These results, compared with previous ones, show an age-related evolution in pathological state in SHR-SP. They point out a specific concentration of the beta-blocking drug in a defined part of the brain, namely medulla + corpus trapezoides.     

Norepinephrine concentration in kidneys of normotensive Wistar-Kyoto and spontaneously hypertensive rats.

Renal norepinephrine (NE) concentration was measured in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) at 7, 9, 11, and 13 weeks of age. Although the weight of kidneys was similar in the two strains of rats, renal NE concentration was significantly lower in SHR at all ages (147 +/- 9 to 175 +/- 13 ng/g for SHR, and 216 +/- 8 to 262 +/- 17 ng/g for WKY rats). The difference in renal NE concentration during this time of rapidly increasing arterial pressure in the SHR suggests that renal NE may in some way be related to the development of hypertension.