Abstract
Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.
Highlights
Alkaline phosphatase (ALP; EC 3.1.3.1.; orthophosphoric-monoester phosphohydrolase, alkaline optimum) is ubiquitous in nature; its physiological role and natural substrates remain largely unknown [1,2]
Atenolol (2 mM) induced a significantly stronger activation (36%) than 2 mM propranolol; propranolol presented a tendency to activate ALP. This is the first report concerning rat heart ALP isoenzyme identification, total ALP activity quantification in the rat heart and its in vitro acute modulation by compounds and polyphenol-rich beverages reported in the literature to have cardiovascular beneficial effects
The inhibitory effects of levamisole and L-phenylalanine showed that both Tn- and tissue-specific ALPs (Ts-ALP) were present in rat heart, since these compounds are, respectively, specific inhibitors of these two ALP groups [2,13,19]
Summary
Alkaline phosphatase (ALP; EC 3.1.3.1.; orthophosphoric-monoester phosphohydrolase, alkaline optimum) is ubiquitous in nature (from bacteria to humans); its physiological role and natural substrates remain largely unknown [1,2]. The degree of aortic valve calcification is a strong predictor of both the progression and the outcome of aortic stenosis. These calcified lesions contain various components associated with bone mineralization, such as ALP, and inflammatory cells such as macrophages and lymphocytes. The mechanisms underlying valve calcification have not been established, recent data suggest that valve calcification is an active process, much like atherosclerosis, that is preceded by inflammation, lipid deposition and the accumulation of extracellular bone matrix proteins [1,6,7,8,9,10,11]
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