One of the earliest cellular responses to prolactin (PRL) binding in Nb2 cells, a rat pre-T lymphoma cell line, is an increase in tyrosine phosphorylation of cellular proteins. In this work, immunologic techniques have been used to demonstrate that in Nb2 cells and in mouse mammary gland explants, JAK2, a non-receptor tyrosine kinase, is activated following stimulation with PRL. PRL stimulated tyrosine phosphorylation of JAK2 at times as early as 30 sec and concentrations of PRL as low as 0.5 ng/ml (2.5 pM) in Nb2 cells and 100 ng/ml (5 nM) in mammary gland explants. When JAK2 was immunoprecipitated from solubilized Nb2 cells or mamMary gland explants and incubated with [r32P]ATP, 32P was incorporated into a protein mirating with an apparent molecular weight appropriate for JAK2 only when cells had been incubated with PRL, indicating that JAK2 tyrosine kinase activity is exquisitely sensitive to PRL. In Nb2 cells, JAK2 was found to associate with PRL receptor irrespective ofwhether or not the cells had been incubated with PRL. These results provide strong evidence that JAK2 is constitutively asiated with the PRL receptor and that it is activated and tyrosine phosphorylated upon PRL binding to the PRL receptor. These results are consistent with JAK2 serving as an early, perhaps initial, sgnaling molecule for PRL.

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.

No abstract

Iodide is an essential constituent of milk that is present in concentrations more than an order of magnitude higher than in the maternal plasma. Earlier, a sodium-iodide symporter was identified in the mammary gland; this transporter is presumed to take iodide from the maternal plasma into the alveolar epithelial cells of the mammary gland. We now report the existence of a second iodide transporter, pendrin, which is also essential for iodide accumulation in milk. Via Western blotting methods, high levels of the transporter were detected in lactating tissues; lesser amounts were found in tissues from midpregnant and virgin mice. Prolactin, at physiological concentrations, stimulated the expression of the pendrin transporter in cultured mammary tissues taken from 12- to 14-day-pregnant mice. The prolactin effect on iodide uptake into cultured mammary tissues was abolished by pendrin transport inhibitors, including DIDS, furosemide, and probenecid. These studies suggest that the prolactin stimulation of pendrin activity is an essential element in the prolactin stimulation of iodide uptake into milk.

We have recently reported that prolactin (PRL) stimulates iodide uptake into cultured mouse mammary tissues. This effect occurs in both TCA soluble and insoluble tissue fractions. The effect of PRL apparently involves an RNA-DNA-dependent mechanism, since actinomycin D and cyclohexamide abolish the PRL stimulation of iodide uptake and its incorporation into protein. Perchlorate and thiocyanate, inhibitors of the iodide transporter, also abolish the PRL effects on iodide uptake and incorporation. Similarly, propylthiouracil and aminotriazole, inhibitors of peroxidase, abolish both effects of PRL. Finally, the extent of iodide uptake in mammary cells is suppressed by about 50% in sodium-free medium. These studies thus suggest the existence of a sodium-iodide symporter in the mammary gland which has characteristics similar to the iodide transporter in the thyroid gland-that is, it is sodium dependent and is inhibited by perchlorate and thiocyanate. The fact that both iodide transporter inhibitors and peroxidase inhibitors abolish PRL-stimulated iodide uptake and incorporation suggests that there may be a coupled mechanism involving the iodide transporter and the peroxidase enzyme.