Processing of rat prolactin by rat tissue explants and serum in vitro

Previous work has shown that enzymes from rat liver or mammary gland can cleave rat (r) PRL to form a two-chain derivative that yields approximately 16- and 7-kilodalton (kDa) fragments upon reduction. Both cleaved rPRL and the purified 16-kDa fragment have maintained biological activity. Thus, cleavage may be of physiological significance. To determine whether rPRL can be cleaved by intact cells, and to evaluate the extent to which rPRL processing is tissue specific and varies with physiological state, rPRL was incubated with slices of different tissues from cycling, midpregnant, or 15-day lactating rats. The molecular mass and relative abundance of rPRL and fragments of the hormone in the medium were determined using reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with an antiserum to the 16-kDa fragment of rPRL. Parenthetically, the fragment that was previously identified as having a molecular mass of 16 kDa had the same mobility on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the form that we designate as having a molecular mass of 14 kDa. Kidney, spleen, mammary gland and liver explants from pregnant rats processed PRL in qualitatively and quantitatively different ways. Mammary gland from lactating rats produced more of a 14-kDa fragment than liver or kidney slices from lactating rats, and the capacity to produce 14-kDa PRL by mammary gland from rats in different physiological states was as follows: pregnant greater than cycling greater than lactating. Fragments of 11 and 16 kDa were also produced, but only by mammary gland from lactating rats, and the latter only after 3 h of incubation. After a 2-h incubation, PRL-like immunoreactivity in lactating mammary gland medium was composed of 23-, 14-, and 11-kDa forms in the following relative amounts: 65 +/- 9, 17 +/- 1, and 10 +/- 5%, respectively. These results suggest that PRL is cleaved in a manner that varies with different tissues and physiological states; and thus, forms are produced that might be important mediators of PRL's biological actions on different target tissues. Medium conditioned by preincubation with mammary gland from lactating rats and clarified by 15,000 x g centrifugation had no PRL-cleaving activity at pH 7.4, but gained activity when the pH was lowered, with maximal activity at pH 2.6-3.0. When heated to 85 C for 15 min, such medium had no activity at pH 3.0.(ABSTRACT TRUNCATED AT 400 WORDS)