Kallikrein hydrolyzes various biologically active peptides, other than kininogens, such as vasoactive intestinal polypeptide (VIP), in vitro. Since kallikrein and VIP have been immunohistochemically shown to be present in the perivascular areas of the pineal gland, this study was designed to determine their topographic proximity in these glands, using immunohistochemical and immunoelectron microscopic double staining methods. Furthermore, since this gland is well-known to have a circadian rhythm, the kallikrein content was measured every 4 h, using a synthetic substrate, Pro-Phe-Arg-MCA, and an enzyme-linked immunosorbent assay (ELISA) to determine whether kallikrein has a circadian rhythm. The immunoreactivities of kallikrein and VIP were highly localized in the perivascular extracellular spaces and were virtually identical in distribution. The kallikrein content changed every 4 h and was high under light and low under dark conditions. The change was more evident when the synthetic substrate was used, and this rhythm was subtle on ELISA. VIP is also said to have a circadian rhythm in the pineal glands, being low under light and high under dark conditions, i.e., opposite to that of kallikrein. Since kallikrein degrades VIP in vitro, it is reasonable to speculate that pineal gland kallikrein is involved in the processing of VIP and possibly other biologically active peptides in the perivascular areas with a discernible circadian rhythm.
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