Distribution of three microvillar enzymes along the small intestinal crypt-villus axis.

Aminopeptidase N (ApN), dipeptidyl peptidase IV (DPP IV) and sucrose-isomaltase (S-I) are differentially expressed along the pig jejunum crypt-villus axis. Quantitative immunoelectron microscopy and enzyme cytochemistry show that DPP IV and S-I are expressed in enterocytes along the entire length of the axis, whereas ApN is found mainly in the villar and upper crypt enterocytes. All three enzymes are detected in the basolateral membrane at all levels along the crypt-villus axis, although ApN and S-I only occurred at low intensities in the villus region. The microvillar/basolateral labelling ratio for the three enzymes increases to a varying degree for the three enzymes along the axis suggesting that the sorting efficiency to the apical membrane improves at least for ApN and S-I as the cells mature. These findings might indicate that the enterocytes change from a transcytotic to a direct apical transport as the enterocytes mature.

On the transfer of serum proteins to the rat intestinal juice.

The in vivo pattern of serum proteins in the rat small-intestinal juice was characterized by crossed immunoelectrophoresis. Immunoglobulins and albumin, alpha-1-antitrypsin, transferrin, and orosomucoid were present. Larger serum proteins were absent (ceruloplasmin, haptoglobin, alpha-1-macroglobulin, alpha and beta lipoproteins). Thus, apart from immunoglobulins, only serum proteins with a molecular mass less than approximately 100 kDa were demonstrated. The origin and epithelial transfer were further characterized, using albumin as a model. No sign of local synthesis of albumin by the enterocytes was found by Northern blotting, and no albumin was found in the Golgi complex by immunogold electron microscopy. By immunogold electron microscopy a heavy labelling of albumin was observed in the interstitial spaces between the villus enterocytes. Where the enterocytes disintegrated, albumin was seen to leak out into the intestinal lumen from the opened interstitial spaces. A weak labelling was also found in the lysosomal/endosomal-like structures, especially in the crypt enterocytes, indicating pinocytosis of albumin. We conclude that the main reason for the occurrence of certain serum proteins in the intestinal juice is a selective passage through the capillary wall followed by passive intercellular transport via delivery of the serum in the interstitial space during disintegration of the enterocytes.

Apical secretion of apolipoproteins from enterocytes.

Synthesis and secretion of apolipoproteins in pig small intestine was studied by pulse-chase labeling of jejunal segments, kept in organ culture. Apo A-1 and apo B-48 were the two major proteins released, constituting 25 and 10%, respectively, of the total amount of labeled protein in the mucosal-side medium where they appeared with a t1/2 of 50-60 min. Using tissue from fasting animals, > 85% of newly synthesized apo A-1 and about one third of apo B-48 was released to the mucosal-side medium. Newly synthesized apolipoprotein that remained associated with the intestinal segment accumulated in the soluble fraction, suggesting a basolateral secretion into the intercellular space, and both this accumulation and the release to the medium was prevented by culture at 20 degrees C. The specific radioactivity of apo A-1 and apo B-48 released to the medium was significantly higher than that of the corresponding apolipoproteins remaining associated with the intestinal tissue. Furthermore, during culture periods of up to 5 h, the enterocytes and their tight junctions largely remained intact as evidenced by the inaccessibility of the nonpermeable surface marker Ruthenium red. We therefore propose that enterocytes release most of their newly made free apo A-1 and a significant portion of apo B-48 by exocytosis via the brush border membrane into the intestinal lumen. Fat absorption reduced apolipoprotein secretion to the medium and induced the formation of chylomicrons, containing apo A-1 at their surface, as evidenced by immunogold electron microscopy. The chylomicrons were localized in the Golgi complex and near the basolateral plasma membrane, but not in the apical region of the enterocytes, indicating that only free apolipoproteins are secreted to the intestinal lumen.

Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells.

Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein, since the gold particles appear all over the granules and are not specifically associated with the granule membrane.