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  • It is reported that sympathetic nerve activity


    It is reported that sympathetic nerve activity is increased in the SHR strain compared with that in the WKY strain (Judy and Farrell, 1979, Lundin et al., 1984, Sugimura et al., 2008) and NA content is increased in the CB of the New Zealand strain of hypertensive rat compared with that in normotensive rat (Pallot and Barer, 1985). These findings suggest that NA release from sympathetic nerve fibers in addition to glomus cells within the CB is increased in SHR/Izm; therefore, it was expected that DBH immunoreactive nerve fiber within the CB is increased in SHR/Izm compared with that Tideglusib in WKY/Izm. In contrast, DBH immunoreactive nerve fibers were at similar levels between the two rat strains in the present study. Because it is known that DBH is released from synaptic vesicles together with NA (Smith et al., 1970, Weinshilboum et al., 1971), it is suggested that DBH immunoreactive nerve fibers are not increased in SHR/Izm.
    Introduction Dopamine is hydroxylated to norepinephrine by the catalytic effect of dopamine β-hydroxylase (DBH), one of the enzymes involved in catecholamine biosynthesis [1]. The enzymatic process involves the oxidation of ascorbic Tideglusib to dehydroascorbate and the reduction of Cu2+ to Cu+ [2]. The enzyme is involved in the regulation of blood pressure by the nervous system and a target for antihypertensive drugs [3]. DBH is situated within catecholamine-containing chromaffin granules and exists in two forms: a membrane-bound form that is reinternalized upon exocytosis and a soluble form that is stored in the granule and secreted [4]. The two states of the enzyme in the chromaffin granules are immunochemically identical; however, they display differences in pH stability and substrate affinity [5]. With the key role that DBH plays in the biosynthesis of catecholamines, this process has been subject of a number of studies. The majority of the assays utilize crude extracts giving rise to the possibility of endogenous inhibitors in the extracts that can interfere with DBH activity [6]. When highly purified enzyme is used it involves long and complicated purification procedures which are costly. The enzymatic assays can also be cumbersome, for example, one assay utilizes a two-step enzyme method, using phenylethanolamine N-methyltransferase and S-adenosyl-l-methionine [6]. However, with these techniques saturating substrate concentrations for DBH cannot be examined due to the inhibition of the second enzyme, phenylethanolamine N-methyltransferase. The main problem faced by researchers when characterizing DBH is the repeated need for large amounts of highly purified enzyme. However, there are recent applications that utilize enzymes in one or more immobilized form [7], [8]. The enzymes were immobilized onto solid supports and used in batch incubations [7], [9] or as biosensors (cf. Refs. [8], [9], [10], [11], [12], [13]). These applications do not require highly purified enzymes and decrease the amounts of enzyme required. The immobilized enzymes retain their activity and can be reused following a simple washing procedure. In this study, the hydrophobic character of the IAM stationary phase was used to immobilize commercially available partially purified dopamine β-hydroxylase. The IAM interphase is derived from the covalent immobilization of 1-myristoyl-2-[(13-carboxyl) tridecanoyl]-sn-3-glycerophospholine on aminopropyl silica, and resembles one-half of a cellular membrane [14]. The hydrocarbon chains create interstitial spaces that allow for the insertion of DBH. DBH was also immobilized onto glutaraldehyde-P (Glut-P), a wide-pore silica that has been covalently clad with polyethyleneimine, a hydrophilic polymer [15]. The reactive amine groups of the polymer form a covalent bond with glutaraldehyde. This particular support is ideal for immobilization of proteins with primary amino groups that form an amine–aldehyde Schiff linkage with Glut-P [15].
    Materials and methods