Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells.

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.

Rates of proximal and distal glycosylation of luteinizing hormone by cultured rat pituitary cells.

The purpose of the present work was to study the rate of basal luteinizing hormone (LH) glycosylation, discriminating the co-translational (proximal) and the post-translational (distal) glycosylation. The experiments were performed to determine the temporal relationship between the biosynthesis of the peptide chains (by [14C]leucine incorporation: [14C]Leu-LH) and the proximal (by [3H]mannose incorporation: [3H]Man-LH) and distal (by [3H]galactose incorporation: [3H]Gal-LH) glycosylation of LH, by rat pituitary cells in primary culture. In addition, the effects of cycloheximide (translation inhibitor) and tunicamycin (glycosylation inhibitor) on the rates of synthesis and release of [3H]Man-LH and [3H]Gal-LH were studied. The rates of synthesis and release (between 0 and 2 h and between 2 and 5 h) were calculated by regression analysis and the statistical significance of differences was determined by the slope test. The rate of synthesis of [3H]Man-LH (slope = 45.59) parallelled that of [14C]Leu-LH (slope = 41.39), which was in agreement with the assumption that the addition of the high mannose core is a co-translational event. Release of [3H]Man-LH (slope = 4.32) as well as that of [14C]Leu-LH (slope = 2.53) showed a lag period of approximately 2 h. The dynamics of [3H]Gal-LH secretion over the course of incubation, with a slower rate of synthesis (slope = 26.40) and a faster rate of release (slope = 6.34), differed from that of [3H]Man-LH. LH labeled with [3H]Gal was released from the early times of the incubation, indicating that galactose is added in the final stages of the secretory process into LH molecules, which are immediately released. LH translation blockage induced by cycloheximide was associated with a corresponding decrease of [3H]Man incorporation. On the other hand, addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH, without affecting those of [14C]Leu-LH. These findings show that proximal glycosylation depends on synthesis of the peptide chains, whereas the addition of the polymannose core is not a condition for translation. The rates of synthesis and release of [3H]Gal-LH were less affected by the antibiotics, and the inhibition was only significant at higher doses and long-time treatments. The present results demonstrate the independence of both steps of LH glycosylation, the rate of [3H]Gal-LH synthesis being 1.7-fold slower and that of release 1.5-fold faster than those of [3H]Man-LH, respectively. The data also suggest that glycosylation is not an essential step in the LH secretory process since the hormone, which is normally secreted in a glycosylated form, was synthesized, transported, and released without the carbohydrate side chains.

Kinetics of translation and glycosylation of pituitary glycoproteins.

The present study determines the basal kinetics of synthesis of translation (by [14C] leucine incorporation, [14C]leu-PROT] and of proximal (by [3H]mannose incorporation, [3H]man-PROT) and distal (by [3H]galactose incorporation, [3H]gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both steps of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM;glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [14C]leu plus [3H]man or [14C]leu plus [3H]gal, for various time-intervals (from 0.5 to 5 h) in the absence (control) or presence of different doses of CH (1.0; 4.0 or 16.0 micrograms/ml) or TM (0.5; 1.0 or 2.0 micrograms/ml). The kinetics of synthesis (slope = 3488) and release (slope = 622) of [14C]le-PROT were higher than those of the sugar precursors (slopes: [3H]man-PROT = 1751 and 526; [3H]gal-PROT = 1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2 h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30 min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [3H]man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [3H]man-PROT without affecting those of [14C]leu-PROT. The kinetics of synthesis and release of [3H]gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell.

Kinetics of translation and glycosylation of pituitary glycoproteins

The present study determines the basal kinectics of synthesis of translation (by[(14)C] leucine incorporation. [(14)C] leu-PROT] and of proximal (by [(3)H] mannose incorparation. [(3)H] man-PROT) and distal (by [3H] galactose incorporation, [(3)H] gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both septs of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM; glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [(14)C] leu plus [(3)H] man or [(14)C] leu plus [(3)H] gal, for various time-intervals (from 0.5 to 5h) in the abscence (control) or presence of different doses od CH (1.0;4.0 or 16.0 mug/ml) or TM (0.5;1.0 or 2.0 mug/ml). The kinetics of synthesis (slope=3488) and release (slope=622) of [(14)C] le-PROT were higler than those of the sugar percursos (slopes: [(3)H] man-PROT=1751 and 526; [(3)H] gal-PROT=1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [(3)H] man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [(3)H] man-PROT without affecting those of [(14)C] leu-PROT. The kinetics of synthesis and release of [(3)H] gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell.

Kinetics of translation and glycosylation of pituitary glycoproteins

The present study determines the basal kinectics of synthesis of translation (by[(14)C] leucine incorporation. [(14)C] leu-PROT] and of proximal (by [(3)H] mannose incorparation. [(3)H] man-PROT) and distal (by [3H] galactose incorporation, [(3)H] gal-PROT) glycosylation of total adenohypophyseal glycoproteins, by rat pituitary cells in primary culture. In order to obtain more information regarding the role of both septs of glycosylation on the secretory process, the effects of cycloheximide (CH; translation inhibitor) and tunicamycin (TM; glycosylation inhibitor) on the kinetics of synthesis and release of pituitary glycoproteins were also studied. Cells were incubated in medium containing [(14)C] leu plus [(3)H] man or [(14)C] leu plus [(3)H] gal, for various time-intervals (from 0.5 to 5h) in the abscence (control) or presence of different doses od CH (1.0;4.0 or 16.0 mug/ml) or TM (0.5;1.0 or 2.0 mug/ml). The kinetics of synthesis (slope=3488) and release (slope=622) of [(14)C] le-PROT were higler than those of the sugar percursos (slopes: [(3)H] man-PROT=1751 and 526; [(3)H] gal-PROT=1231 and 506). Leucine or mannose-labeled protein was barely detectable in the medium after 2h incubation, whereas galactose-labeled protein had already been released into the incubation medium by 30min. Cycloheximide induced translation blockage and, concomitantly, produced a marked inhibition of [(3)H] man incorporation. On the other hand, TM inhibited the kinetics of synthesis and release of [(3)H] man-PROT without affecting those of [(14)C] leu-PROT. The kinetics of synthesis and release of [(3)H] gal-PROT, although diminished, maintained linearity and increased in function of time, even in the presence of the antibiotics. Thus, the present results on glycoproteins from the pituitary gland are consistent with the previous conclusion for other mammalian glycoproteins that carbohydrate attachment occurs in several steps to molecules destined to be secreted. Addition of mannose (proximal glycosylation) is a co-translational event and that of galactose (distal glycosylation) is post-translational and can be designated as final stages in carbohydrate assembly, occurring close to the time of release. Furthermore, it has been demonstrated that the absence of the carbohydrate side chains of the pituitary glycoprotein does not prevent the intracellular transport of the protein and its export from the cell. (AU)