Changes in molecular variants during in vitro transformation and release of prolactin by the pituitary gland of the lactating rat

Abstract

PRL transformation involves a dopamine (DA)-controlled, thiol-mediated decrease in pituitary PRL detectability that precedes and may determine increased PRL release. The present studies were designed to determine 1) whether structural changes occur to adenohypophyseal (AP) PRL during in vitro transformation and may account for decreased PRL detectability; 2) whether such changes occur within AP PRL granules; 3) the type and proportion of secreted PRL variants; and 4) the influence of DA and/or thiol reducing agents upon AP PRL molecular changes. Quartered APs of 8 h nonsuckled lactating rats, injected or not with [3H]leucine (5 mCi/g body wt, 8 h before death) were incubated for 30-240 min. The effects of DA (50 μM) with or without reduced glutathione (10 mM), and the alkylant N-ethylmaleimide (1 mM) were compared to the changes in PRL molecular variants occurring in control incubated AP fragments. Polyacrylamide gel electrophoresis (PAGE) under native conditions and sodium dodecyl sulfate (SDS)-PAGE under reducing (R) or nonreducing (NR) conditions were used to resolve molecular variants of PRL and [3H]PRL, followed by Western blotting and densitometric/liquid scintillation techniques, respectively. Up to 5 and 10 major forms of immunoreactive PRL were resolved by PAGE and SDS-PAGE, respectively. The spontaneous transformation after 30 min incubation was correlated with a decrease of 23K PRL and 23K [3H]PRL bands and an increase in high mol wt (80 to >100K) PRL forms. These changes were reversible upon reduction of the proteins before electrophoresis or after extraction of preparative gels. DA and N-ethylmaleimide prevented, whereas reduced glutathione induced these changes. Similar changes were observed in isolated granules from nonincubated and incubated pituitary fragments. Secreted PRL was also polymorphic, with a high predominance of 23K PRL and 23K [3H]PRL. In addition, a polymeric PRL variant, which in reducing gels showed an apparent mol wt of 27K, was secreted in high proportion, similar to that of 23K PRL. The nature of this polymeric variant of PRL is unclear at present. Altogether, these results suggest that a dynamic interconversion of PRL molecular variants occur during transformation. The decresed detectability associated with PRL transformation appears to involve the association of 23K PRL molecules into a 80 to >100K disulfide-linked oligomer. This association is reversible upon reduction and takes place within secretory granules. DA appears to inhibit PRL transformation by preventing thiol-disulfide interchange.