Reaction Sites by Cerium-Cytochemistry for the Hydrogen-Peroxide Generating Enzymes in the Mongolian Gerbil Parotid Gland.

Abstract

In order to cytochemically demonstrate the endogenous glandular source of hydrogen-peroxide (H2O2), which is a component of the salivary peroxidase antimicrobial system, we applied the cerium method to the Mongolian gerbil parotid gland. When NADH and NADPH were used as substrates of the H2O2 generating enzyme, reaction products were observed in association with the plasma membrane and pinocytotic vesicles of the myoepithelial cell. No reaction product was formed in the absence of these substrates. The reaction was inhibited by the addition to the complete medium of catalase (H2O2 scavenger) or of p-benzoquinone (O2-scavenger), or by heating prior to incubation. These results suggest that NADH-and NADPH-dependent H2O2 generating oxidase may be present in the myoepithelial cell. In addition, electron-dense precipitates, formed by the reaction of cerous ions, were also observed on the outer surface of the luminar plasma membrane of acinar and intercalated duct cells, and in the cristae and outer compartment of mitochondria mainly of duct cells. However, these reaction products were formed whether the substrates were contained in the cerium medium or not. Since the reaction was also weakened by catalase or p-benzoquinone, or by preheating, it is likely that some endogenous H2O2-generating factors, independent bf the exogenous substrates, may exist in these sites.

Cytochemical Localization of NAD(P)H Oxidase in the Myoepithelial Cells of Salivary and Other Exocrine Glands.

“…The reactions associated with MEC were, in principle, the same as those reported in our previous study on the Mongolian gerbil parotid gland [13], and suggest that some H202-generating system by NAD(P)H oxidase may be present in association with the MEC of the mammalian exocrine glands. So far there has been no information about the functional significance of the H2O2 generation system in MEC.…”

“…Reactivities of NAD(P)H oxidase cytochemistry on myoepithelial cells (MEC) in several mammalian exocrine glands 1) Reactivity is expressed by the amount and continuity of deposits in a NAD(P)H oxidase positive MEC: heavy and continuous, ++; visible and continuous, +; visible but irregular, ± 2) Universality of reactions on different MEC in the same gland is expressed as follows: almost all the MEC are reactive, ++; some MEC are nonreactive, +; few MEC are reactive, ± 3) Data from our previous report [13] differentiation on each side of the membrane. The distribution of activity in MEC, which is densely associated with the neighboring cells, implies that NAD(P)H oxidase is involved in some intercellular regulatory system.…”

“…The tissues were immediately fixed for 1 hr in a cold 1% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4. NAD(P)H oxidase cytochemistry was performed using a previously described cerium method [13]. Some control incubations were performed as follows: (1) incubation in a substrate-free medium; (2) incubation in a complete medium containing 10 mg/ml catalase (bovine liver, C-40, Sigma), a scavenger of H2O2; and (3) heating for 5 min at 65 ° C prior to the incubation.…”

“…In the thyroid follicular cells, NADPH oxidase is known to be involved in the biosynthesis of thyroid hormone [5]. We found in a previous study about the H2O2 generating sites in the parotid gland of the Mongolian gerbil that NAD(P)H oxidase could be exclusively localized on the myoepithelial cells (MEC) in the parenchyma of the gland [13]. Until then, in the MEC of exocrine glands, there had been no report demonstrating the localization of NAD(P)H oxidase.…”