In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms.
The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione; for example, 180 min values were 156% (GH) and 439% (PRL) at 22 C compared with control homogenate values. Under these conditions, PRL in granule cores (the sedimentable fraction after hypotonic lysis of granules) increased the most, changing 120-fold from 2.9 +/- 0.25 micrograms/ml (pH 8.3 control) to 349 +/- 22.8 micrograms/ml. Isolated oligomeric PRL increased significantly after exposure to mercaptoethanol, indicating that changes in assayability do not require nonhormonal granule components. No change in monomeric PRL, or GH in either form, was observed. Pretreatment with iodoacetamide resulted in a 33% decrease in maximal PRL values, indicating the presence and functional importance of granule thiols. Cysteamine inhibition of bPRL values in cores was modulated by urea, EDTA, and iodoacetamide. Though 5 M urea or 0.5% sodium dodecyl sulfate (SDS) increased granule PRL at pH 8.3, maximal values required thiols at pH 10.5. In contrast, maximal GH values were obtained with SDS at pH 8.3 without thiols. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion.
D000818:Animals; D001696:Biomechanical Phenomena; D002417:Cattle; D003543:Cysteamine / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000032:analysis / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D013006:Growth Hormone / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D010902:Pituitary Gland / Q000032:analysis* / Q000378:metabolism; D011388:Prolactin / Q000032:analysis* / Q000378:metabolism; D011863:Radioimmunoassay; D013696:Temperature; D013997:Time Factors
D003543:Cysteamine; D011388:Prolactin; D013006:Growth Hormone; D004492:Edetic Acid; D005978:Glutathione