Histochemistry of 11-beta-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation.

Abstract

The activity pattern of NAD/NADP-linked 11β-hydroxysteroid dehydrogenase (HSD) in the submandibular gland of the rat was re-evaluated using several control experiments. The incubation time needed for the initial appearance of red and blue formazans was used to investigate the activity of NAD-dependent 11β-HSD in control and cortisol-treated rats. The following results were obtained. (1) Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH 7.2) for up to 20 min preserved morphological integrity and maximal enzyme activity. The substantivity of formazans was enhanced. (2) The substantivity of Nitro BT was highly variable. The implication of this forin situ localization of enzymes was analysed. (3) Pretreatment with acetone and application of phenanthroline was necessary to avoid a false positive reaction caused by alcohol dehydrogenase. (4) No diffusion of 11β-HSD was noticed within 30 min of incubation, nor was rediffusion of reduced intermediates seen. (5) With either NAD or NADP as coenzyme, 11β-HSD was localized in the striated, intralobular, interlobular, interlobar, and main duct. (6) 11β-HSD was found to be primarily NAD-dependent. (7) With DMF or DMSO as solvent, the rate of utilization of substrates was as follows: Cortisol=11β-hydroxyandrostendione>11β-hydroxyprogesterone. Aldosterone was utilized very poorly, if at all. (8) After injection (i.p.) of a single pharmacological dose of cortisol, the activity of NAD-linked 11β-HSD was significantly increased 24 h later. (9) NADH-tetrazolium reductase was not inhibited by levamisole. (10) Distinct NADPH-tetrazolium reductase activity was localized in the apical part of cells (or cell membranes) of the interlobar ducts and the main duct.