Three aspects of the secretory process in male rat prolactin (PRL) cells grown in primary cultures for 7--14 days have been investigated by cytochemical methods. The subcellular localization of prolactin has been studied using preembedding or postembedding immunocytochemical methods after various fixatives. With the postembedding method, PRL is localized essentially in secretory granules. The maximum intensity of staining is obtained with PAF fixative. With the preembedding method, subcellular localization of the staining varies depending on the fixative. After PAF-fixation, positive staining is observed on secretory granules, ground cytoplasm, the outer face of some RER cisternae and, in a few cells, on the innermost Golgi cisternae, as well as on masses of condensing secretory material. After Ohtsuki's hypotonic fixative followed by saponin permeabilization, PRL is visualized within the totality of RER cisternae, including the perinuclear cisternae and the peripheral saccules on the cis-Golgi face. Secretory granules are unstained. Membrane traffic was investigated using the Con A-HRP indirect method as a tracer of surface saccharides. Plasma membrane, coated with Con A-HRP at 4 degrees C, is slowly internalized at 37 degrees C. This involves both randomly distributed invaginations and capping. The final step of endocytosis (1--2 hours) is located in the Golgi zone, where very few smooth membranes are stained. In contrast, a conspicuous deposit is found around the dense content of secretory granules. This suggests a recycling of internalized membrane and a transfer of Con A-HRP from the inner face of smooth cisternae to the secretory material. The internalization of Con A-HRP-coated membrane leads to an inhibition of PRL release starting after 30 minutes. This is accompanied by a marked increase of acid phosphatase activity, mostly around forming and mature secretory granules.
(6A0727). trope, thyrotrope, and corticotrepe cells but were very scarce in prolactin cells and absent in somatotrope cells. In features or by immunodetection of its hormone content. These findings suggest that laminin might be synthesized and exported by all the glandular anterior pituitary cells, according to different pathways. Materials and Methods Materials Antibodies. Affinity-purified goat antibodies to laminin (a.laminin)
We have isolated a full-length murine clone corresponding to the rat neuronal p1A75 partial cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E. (1983) Cell 33, 671-682). It encodes a 185-residue polypeptide that displays 56% identity with p19, a protein selectively expressed in the Golgi apparatus of neural cells (Sabé ran-Djoneidi, D., Marey-Semper, I., Picart, R., Studler, J.-M., Tougard, C., Glowinski, J., and Lé viStrauss, M. (1995) J. Biol. Chem. 270, 1888 -1893). An antibody directed against the recombinant polypeptide allowed us to demonstrate the existence of the natural 21-kDa protein (p21) in brain and its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural observation of cultured neurons and analysis of transfected COS cells revealed a specific labeling of the Golgi apparatus, suggesting, as for p19, the presence of a Golgi targeting signal in its primary sequence. Surprisingly, p21, which is much more strongly expressed in the olfactory epithelium than p19, is also present in the Golgi complex of spermatocytes and in the flagellar middle piece of late spermatids.We have previously described a 19-kDa murine protein (p19) selectively expressed in the Golgi apparatus of neural and neuroendocrine cells whose human corresponding gene has been localized in 5q35 (1, 2). The primary sequence of p19 showed a 57% similarity with the translation product of an open reading frame of the rat neuronal p1A75 (3) partial cDNA, which was isolated 14 years ago and whose human corresponding gene is localized in 4p16 (4). Searches in protein data bases for other members of this new family have only revealed that these two proteins share a highly similar short segment with secretogranin III, which is expressed in intracellular vesicles of neural cells (1, 5).The co-localization, in two paralogous chromosomal regions (5q35 and 4p16), of the human p19 and p1A75 genes with other couples of homologous genes such as, for instance, the D1 and D5 dopamine receptors and the FGFR3 and FGFR4 fibroblast growth factor receptors suggested that these genes originated from the same large gene duplication event, which is thought to be the remnant of an ancient round of tetraploidization (6).To characterize this new protein family and to study the functional consequences of a well defined large duplication event, we undertook the thorough analysis of the protein encoded by the p1A75 cDNA.This encoded protein is a 21-kDa protein (p21) that is expressed, like p19, in the Golgi apparatus of neural and neuroendocrine cells. However, unlike p19, which is absent from the testis and faintly expressed in the olfactory epithelium, p21 is very strongly expressed in the olfactory epithelium and in male germ cells. EXPERIMENTAL PROCEDURESRNA Isolation and Northern Blotting-Total cellular RNA was extracted from fresh tissue or cells by the guanidium thiocyanate/phenol chloroform extraction method (7). Timed pregnant OFA rats (IffaCredo) provided a source of fetal and neonatal brains of precise gesta...
The mouse 8.5 mRNA encodes a 171-residue novel protein which displays a highly significant similarity with the product of the previously characterized neuronal p1A75 cDNA (Sutcliffe, J.G., Milner, R.J., Shinnick, T.M., and Bloom, F.E. (1983) Cell 33, 671-682). Northern blot and in situ hybridization experiments indicated that the 8.5 mRNA is specifically expressed in neural and neuroendocrine tissues. An affinity-purified antibody directed against the recombinant 8.5 protein demonstrated the existence of the 19-kDa natural protein in brain and evidenced its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural analysis of the same preparation revealed a specific labeling of all the Golgi saccules and of some vesicles in the Golgi zone. In transfected COS cells, the exogenous protein was also detected in the Golgi area, indicating, therefore, the presence of a Golgi targeting signal in its primary sequence.
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