Spatial and Temporal Differentiation of Alkaline Phosphatase on the Intestinal Villi of the Mouse

The villi of the small intestine are clothed by a constantly renewed population of epithelial cells that are produced in the crypts and extruded at the villi tips (14, 15). During its life of about 1~ days, each epithelial cell undergoes differentiative changes that include a lengthening and narrowing of its microvilli, with the result that the free surface ol apical ceils is very much greater than that of those cells near the bases of the villi (1). Alkaline phosphatase is bound to the membranous coat of the microvilli (5, 8, 12). In the mouse at about 16-18 days of age, the microvilli shift from the short, broad configuration of infancy to the long, narrow, mature form (23), and at the same time the phosphatase activity of the duodenum rises about 20-fold (17). This indication that the increase of phosphatase activity may be related to the expansion of the microvillar membrane, rather than de novo synthesis of enzyme, is supported by the finding that actinomycin D, puromycin, and other drugs that interfere with the synthesis of RNA or protein bring about an elevation of phosphatase activity not only in developmental stages, when phosphatase normally rises (19), but also in adults, when a constant level ordinarily is maintained (20). Since puromycin exerts an activity-enhancing effect under conditions of complete suppression of mitosis, the elevation of phosphatase activity does not depend on the production of new cells (20). The rise of phosphatase activity that occurs in the juvenile mouse is accompanied by a change of characteristics most readily expressed as rate of hydrolysis of phenylphosphate (PhP) relative to that of beta glycerophosphate (bGP) (18). At 11 days this PhP/bGP ratio is below 0.8, at 20 days above 3.0. The change reflects the appearance of two new isozymes, one having a ratio of about 6.0, the other a ratio of approximately 2.0 (21); both continue to be present in the duodenum in adult life. It has been proposed recently that, as cells glide up the villi, the configuration of their microvilli is altered by a mechanism that simultaneously converts the bound phosphatase from one isozymic form to another (20). Presumably the rate at which this chemo-structural differentiation goes forward is regulated by a labile protein, the phosphatase-enhancing effect of actinomycin D or puromycin being to eliminate the restraining effect of this postulated regulator. This hypothesis predicts that the phosphatase isozyme of higher PhP/bGP ratio will be found at the villi tips, and the isozyme of lower ratio at the villi bases. We now have been able to demonstrate the correctness of this hypothesis by means of a technique, recently introduced by Dahlquist and