Guanylin, a bioactive peptide, has recently been isolated from the intet; this peptide activates intestinal guanylate cydase (i.e., guanylate cyclase C) and thus is poten-

Recent studies have established that pinealocytes of the mammalian pineal gland contain marker molecules of neuroendocrine cells or paraneurons like the synaptic vesicle-associated protein synaptophysin (p38). The objective of this study was to identify the subcellular synaptophysin-positive compartment and to characterize in detail the intracellular distribution of this protein in rat and gerbil pinealocytes. An analysis of serial semithin sections of plastic-embedded pineals immunostained for synaptophysin, including computer-assisted optical density measurements of synaptophysin immunoreactivities, demonstrated unequivocally that synaptophysin was highly concentrated in dilated process terminals of the pinealocytes. More than 75% of these process terminals were found to border or lie within the pericapillary space. At the ultrastructural level, they contained accumulations of small clear vesicles of variable size that turned out to be the site of synaptophysin immunoreactivity when immunogold staining was performed. In addition, microvesicles surrounding synaptic ribbons were also immunolabeled. Hence, the pinealocyte is the first neuroendocrine cell type that has now been shown to concentrate synaptophysin-positive microvesicles in perivascular process endings. This observation lends strong support to the hypothesis that small clear vesicles in neuroendocrine cells in general, and in pinealocytes in particular, serve secretory functions. The quantitative analysis of completely sectioned process endings revealed that the microvesicles outnumber by far the amount of dense core vesicles and therefore cannot arise by endocytosis of dense core vesicle membranes. Thus, small synaptic-like vesicles probably constitute an independent secretory pathway of the paraneuronal pinealocytes. In the present study, we could also establish the absence of immunoreactivity for synapsin I (belonging to a family of neuron-specific nerve terminal phosphoproteins) from pinealocytes. Synapsin I immunoreactivity was only detectable in intrapineal nerve terminals and varicosities. Taken together, the immunostaining patterns of the pineal gland obtained with antibodies directed against synaptic vesicle-associated proteins render the mammalian pinealocyte a very special type of neuroendocrine cell or paraneuron rather than a "classic" neuron.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

The study of function and regulation of the phenotype of alveolar type I (AT I) epithelial cells is limited by the rareness of suitable cell lines or primary cultures of this cell type. We describe in the present study the type I-like rat epithelial cell line R3/1. This cell line displays in vitro a phenotype with several characteristic features of AT I cells. R3/1 cells were analysed for mRNA and protein content of markers related to the AT I cell type (T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) and AT II phenotypes [surfactant proteins (SPs) A, B, C and D]. The mRNAs for SPs were found to be at a low level. Moderate protein levels for SP-A and SP-B were found, and SP-C and SP-D proteins were not detectable. R3/1 cells are positive for CD44s, E-cadherin, cytokeratin, vimentin and RAGE, and bind the lectins BPA and SBA. For demonstration of the suitability of R3/1 cells for in vitro studies on epithelial injury, the cells were treated with bleomycin. As shown by real-time RT-PCR and immunoblotting, bleomycin-treatment of R3/1 cells resulted in a decrease in mRNA and protein for both caveolin-1 and caveolin-2 in comparison with controls. The AT I-like cell line R3/1 may serve as a promising tool for the study of lung cell biology.

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety ofendocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in 13 cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (a) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffm cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas ofnormal and pathological conditions may indicate that CST is involved in 13-cell function.Chromogranin A (CGA) is an acidic glycoprotein originally detected in the adrenal medulla but more recently found also in a variety of peptide hormone-producing cells (see refs. 1 and 2 for review). Despite its widespread distribution, the physiological function of CGA is still largely unknown.The primary amino acid sequence of CGA, elucidated by cDNA analysis, includes several pairs of basic residues, which by analogy with other peptide hormone precursors constitute potential sites of proteolytic cleavage (3-5). Thus, it was speculated that CGA may be a putative precursor of biologically active peptides (2, 6, 7). Pancreastatin (PST), a peptide fully contained in the porcine CGA sequence (3, 7), was isolated from the porcine pancreas (8). This peptide inhibits insulin secretion (8). Moreover, tryptic digestion of CGA yielded various peptides that were able to modulate catecholamine secretion (7). Recently, chromostatin (CST) has been identified as a CGA derivative, which is localized between amino acids 124 and 143 in the bovine CGA sequence (9). In contrast to PST, CST had potent inhibitory action on chromaffin cell secretion (9).Previous studies demonstrated that pancreatic islet cells and islet cell tumors contain various CGA-derived peptides (10)(11)(12)(13)(14)(15)(16)(17). In the human pancreas, CGA has been localized mainly to glucagon (a) cells (18). The cellular distribution of PST, however, is still a matter of controversy (see ref. 19 for review), which in part may be due to species specificities (20). Data on tissue distribution of CST are completely lacking.Using specific antisera against CST, PST, and CGA, we inves...