Rat prolactin synthesis by lactating mammary epithelial cells (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
We have previously shown that rat prolactin is proteolyticaily cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically, By modifying an established analytical method, we were able to generate large (/tg) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with 12SI-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50-60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1986
The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.
Clinical Endocrinology, 1977
Pretreatment of female Wistar rats with various compounds (coumarin, 4-methylcoumarin, phenobarbital, carbon tetrachloride) on mammary tumour production by 7,12-dimethylbenz(a)anthracene (DMBA) has been studied. The test compouqds brought about a variety of actions on DMBA tumours; their effect varied from none (phenobarbital, carbon tetrachloride) to decreased incidence and delayed appearance (cournarin, 4-methylcoumarin). In all cases studied increased prolactin level was found. The inhibitory action of coumarin on turnour incidence and multiplicity showed a dose-related association with elevated serum prolactin and reduced hepatic drug metabolism. These results indicate that the production of DMBA-induced mammary adenocarcinomas in the rat is not associated with serum levels of prolactin. Our results suggest that in this model for tumorigenesis, prolactin only plays the role of a promoter after the carcinogenic event.
Cell and Tissue Research, 2011
Despite the important role of prolactin (PRL) in mammary gland development and function, little is known about the distribution of the different forms of the prolactin receptor (PRLR) under various physiological circumstances. Here, the distribution of the long (LF) and the short (S3 in mouse) receptor common to both mice and rats was determined by immunofluorescence on frozen sections of virgin, pregnant and lactating mouse mammary gland. Myoepithelial cells were consistently and intensely stained for both receptors. For luminal cells at all stages (ducts and alveoli), a large proportion of PRLR staining was unexpectedly present on the apical face. In the non-lactating state, no basal staining of luminal cells was detectable. During lactation, a proportion of both receptors moved to the basolateral surface. In vitro, HC11 cells showed constitutive expression of LF but expression of S3 only upon the formation of adherent junctions. Tight junction formation was accelerated by incubation in pseudo-phosphorylated PRL, as measured by transepithelial resistance and the expression and placement of the tight junction protein, zonula occludens-1. Once an intact monolayer had formed, all LF and S3 receptors were apical (akin to the non-lactating state) and only apical application of PRL activated the Jak2-STAT5 and ERK pathways. By contrast, basolateral application of PRL resulted in a reduction in basal ERK phosphorylation, suggesting an involvement of a dual specificity protein phosphatase. Normal human breast samples also showed apical PRLRs. These results demonstrate important contextual aspects of PRL-PRLR interactions with implications for the analysis of the role of PRL in breast cancer.
Isolation, Characterization, and Regulation of the Prolactin Receptor
Journal of Dairy Science, 1985
The prolactin, or lactogenic hormone, receptor has been purified (~ 80%) from lactating mouse liver and human term placenta by the nondenaturing zwitterionic detergent 3-[ (3-cholamidopropyl) -dimethylammonio] -1-propane sulfonate and a prolaetin affinity column. The isolated "core-binding unit" has a molecular weight of 37,000 + 2,000 daltons. It retains the specificity for lactogenic hormones and binds prolactin with an affinity (K a = 2 to 6 × 109M -1) similar to that of the receptor as it occurs in its membranous environment (K a = 3 to 5 × 109M-1). Whether this "core-binding unit" exists on the cell surface in a cryptic or active form is influenced greatly by its association with other membrane proteins and the concentration of phosphatidylcholine within its local membranous environment.
Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats
Annals of Anatomy - Anatomischer Anzeiger, 2003
Lactation is a physiological process associated with hyperactivity of hypophyseal prolactin-producing cells. It is known that the percentage of these cells is increased during lactation, although there are discrepancies in the reports regarding the mechanisms responsible for increasing the number of prolactin cells. In order to analyse whether this increase is a result of previous proliferation, variations in the proliferation rate of prolactin-positive cells were determined from late pregnancy to lactation in adult female rats by means of observation of the immunohistochemical expression of PCNA as a marker of cellular proliferation. During late pregnancy, a very significant increase in the percentage of proliferating prolactin cells was observed in comparison to non-pregnant females in the proestrus phase (p<0.01). Although the percentage of prolactin-positive cells after one week of lactation was