Biosynthesis of the secreted 24 K isoforms of prolactin.

PRL exists within the mammotroph population in a number of different molecular forms. Several of these forms are best described as isoforms, as they have the same mol wt (24 K), but differ in their net charges. In this study we have used in vitro translation assays to ascertain the number of 24 K translation products of normal pituitary messenger RNA (mRNA), and, finding only one, have used both in vitro translation assays and subcellular fractionation to determine the intracellular site of the posttranslational modification of this single translation product. Translation of mRNA from normal pituitary tissue or GH3 cells resulted in the apparent production of a number of pre-PRLs, but in only a single rough microsome-processed form of PRL, 24 K isoform 2. Longer term translation assays utilizing a variety of isotopes failed to show any evidence for rough microsomal posttranslational modification of isoform 2. Subcellular fractionation, using a discontinuous sucrose gradient, however, produced a membrane-bound large secretory granule fraction which, when isolated, contained essentially only isoform 2, and which had the capacity to convert isoform 2 to isoforms 3 and 3' by posttranslational phosphorylation.

Mammotroph autoregulation: the differential roles of the 24K isoforms of prolactin.

In this study we have attempted to determine which of the secreted 24K isoforms was responsible for autocrine regulation of PRL secretion by comparing the isoforms synthesized and secreted by normal cells, which do autoregulate, with those synthesized and secreted by GH3 cells, which do not normally autoregulate. Comparable numbers of cells were washed free of serum and then extracted into Tris-buffered saline by sonication and detergent treatment. Proteins present in these cell extracts and in samples of culture medium were then precipitated with cold acetone (-20 C; 48 h) and subsequently dissolved in urea-lysis buffer for 2-dimensional (2-D) electrophoresis. The 2-D patterns for normal cells showed four 24K PRL isoforms inside the cells and three 24K PRL isoforms (designated 2, 3, and 3') secreted into the medium. The 2-D patterns for GH3 cells showed very little intracellular storage of PRL, but what was present was identified as 24K PRL isoform 2. The GH3 cells secreted large amounts of only 24K PRL isoform 2. Preparations of PRL containing only isoforms 1,2, and 3 (at a total radioimmunoassayable concentration of 5 micrograms/ml PRL) were capable of inducing autoregulation in GH3 cells, as evidence by decreased secretion of prelabeled intracellular PRL. Initiation of autoregulation in GH3 cells caused granulation and the intracellular production of isoform 3. Since a) a preparation containing isoforms 1, 2, and 3 was found to induce autoregulation in GH3 cells, b) isoform 1 is not a secreted form, and c) isoform 2 does not cause autoregulation (at least in GH3 cells), it is deduced that isoform 3 is an autocrine form of PRL. Since initiation of autoregulation in GH3 cells caused those cells to produce isoform 3, it is further deduced that the autoregulatory defect in GH3 cells lies in the actual lack of production of isoform 3 and not in an inherent inability of these cells to produce isoform 3.

Production and secretion of the 21-23.5 kDa prolactin-like molecules.

We recently described the presence of a series of prolactin (PRL)-like molecules (PLMs) in the rat pituitary gland and showed that their formation was not due to artifactual proteolysis of 24 kDa PRL during extraction or to degradation of PRL in lysosomes. In this study we have found (1) in vitro translation of pituitary cell RNA to result in the production of only 24 kDa monomer isoform 2 and no PLMs, (2) that secretion of newly synthesized PLMs is differently regulated than at least a proportion of newly synthesized monomers, (3) that secretion of newly synthesized PLMs occurs after at least a 6 h delay, (4) that cysteamine (a) inhibits the release of the PLMs, (b) causes an increase in their amount versus isoform 2, and (c) causes an intracellular accumulation of pleiomorphic, immature secretory granules, and (5) that cells grown under degranulating culture conditions do not contain PLMs. These results, using normal anterior pituitary cells in primary culture, demonstrate the potential for differential release of the PLMs versus monomer PRL in vivo and are consistent with the production of the PLMs from 24 kDa monomer isoform 2 during secretory granule condensation.

Immunofluorescent and immunoelectron microscopic localization of protein antigens in red cells infected with the human malaria Plasmodium falciparum.

Erythrocytes infected with the human malaria Plasmodium falciparum produce elevations of the surface membrane of the red cell called knobs. Through the use of transmission electron microscopy and a post-embedding protein A-immunogold technique, it was possible to show changes in the distribution of band 3, glycophorin A and spectrin in the region of the knob. These proteins appeared to be aggregated or condensed in the area of the knob, whereas the remainder of the red cell surface showed no such dense clusters; haemoglobin and the histidine-rich protein of P. lophurae could not be localized to the knobby protuberances. It was not possible to detect any changes in protein distribution using the light microscope and indirect immunofluorescence.

Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum.

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.

Plasmodium falciparum: regional differences in lectin and cationized ferritin binding to the surface of the malaria-infected human erythrocyte.

The distribution of anionic residues on the surface of erythrocytes infected with Plasmodium falciparum was studied using cationized ferritin (CF) and transmission electron microscopy. CF staining of uninfected erythrocytes or erythrocytes infected with a knobless variant resulted in a dense and uniform distribution of ferritin particles; however, when red cells infected with a knob-inducing variant were exposed to CF, aggregates of ferritin particles were observed in the region of membrane elevation. Lectin binding to the erythrocyte surface was visualized by transmission electron microscopy using ferritin-conjugated lectins and lectin-fetuin-gold. No differences were observed in the lectin-binding patterns of malaria-infected or uninfected erythrocytes using WGA (wheat-germ agglutinin), RCA (ricin), and Limax flavus lectin. In distinct contrast to the uniform distribution of ferritin particles seen with these lectins was the appearance of clusters of ferritin-ConA over the knobby regions. Localized aggregates of ConA were not seen in knob-free areas or on the surface of red cells infected with a knobless variant. No significant differences were found in the agglutination reactions of normal and infected cells with the Cancer antennarius lectin specific for O-acylated sialic acids.

Receptor mediated action without receptor occupancy.

The relationship between occupancy of a specific cell surface receptor and actions initiated in the same cell by that event was tested on ovarian granulosa cells exposed to hCG. Hormone bound to its receptor was identified by immunocytochemistry or by autoradiography and the dissociation of cAMP-dependent protein kinase was followed by direct cytochemistry. Only 25-30% of the granulosa cells bound hCG and in each instance this resulted in protein kinase dissociation. Cells that did not bind hCG nevertheless dissociated protein kinase if they contacted a cell that had bound hormone and dissociated enzyme. Cells that neither bound hormone, nor contacted cells that did so, failed to dissociate protein kinase. These observations establish, in individual cells, a direct relationship of receptor occupancy to receptor-mediated action and indicate that this event can be communicated to receptorless cells, presumably by gap junctions, thereby amplifying the response to hormone. Similar processes may occur in other tissues wherein receptor-bearing cells are capable of intercellular communication.

Altered red cell membrane fluidity during schizogonic development of malarial parasites (Plasmodium falciparum and P. lophurae).

The plasma membranes of human or duckling erythrocytes infected with malarial parasites (Plasmodium falciparum and P. lophurae respectively) were stained by the fluorescent dye merocyanine 540 in the presence of serum. Unparasitized erythrocytes from infected ducklings or from in vitro cultures remained unstained in the presence of serum. Because merocyanine 540 has a greater affinity for fluid phased or disordered lipid bilayers the results suggest that upon infection of the red blood cell the erythrocyte plasma membrane becomes disordered or is increased in its fluidity. Such alterations of the host erythrocyte are probably due to parasite-induced modifications in the underlying spectrin network (required for lipid leaflet asymmetry) as well as changes in erythrocyte membrane lipid composition.