RESUMO
There is still only limited understanding of the early steps of prolactin (PRL) signal transduction in target cells. Recent studies have identified some of the essential first steps: these include the rapid association of the PRL receptor with JAK tyrosine kinases and tyrosine phosphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the other hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channels involved have not been clearly established. This work was undertaken to characterise the channels responsible for calcium influx and to obtain an insight into their activation processes. Using the patch-clamp technique in the cell-attached configuration, single Ca2+ channel currents were recorded following PRL application (10 nM) in Chinese hamster ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statistical analysis showed that the recorded currents were voltage-independent, with a slope conductance of 16 pS. Although these channels were present in excised patches, the fact that PRL was unable to activate them suggested that a soluble cytoplasmic component may be required. Application of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 microM), to the inside of the excised patch membrane activated the voltage-independent 16 pS Ca2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,4,5)P5 and Ins(1,4,5)P3 did not affect channel activity while InsP6 (20 microM) had some effect, although less marked than that of Ins(1,3,4,5)P4. Using the anion-exchange HPLC technique, we then studied the effects of PRL (10 nM) on the turnover of inositol phosphates (InsPs) in CHO-E32. Our studies showed that PRL induces rapid increases in the production of Ins(1,3,4,5)P4 (207% at 30 s), InsP5 (171% at 30 s), and InsP6 (241% at 30 s). Conversely, Ins(1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomitant increase in Ins(1,3,4,5)P4, suggesting that the former could be transiently phosphorylated to produce the latter. Comparison of the production kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)P4, InsP5, and InsP6 indicated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca2+ entry through voltage-independent Ca2+ channels that may be activated by Ins(1,3,4,5)P4 and InsP6.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Prolactina/farmacologia , Receptores da Prolactina/genética , Animais , Células CHO , Canais de Cálcio/fisiologia , Cricetinae , Cricetulus , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacologia , Potenciais da Membrana/efeitos dos fármacosRESUMO
PRL receptor (PRL-R) activation by PRL triggers a cascade of intracellular events including homodimerization of the receptor, activation of cytoplasmic receptor-associated tyrosine kinase and tyrosine-phosphorylation of various signal transducers. In CHO cells, transfected with the long form of PRL-R, an increase in [Ca2+]i was observed following PRL stimulation whereas Ca2+ is generally coupled with the phosphoinositide metabolism. In this study, we investigated phosphoinositide involvement in the PRL transduction pathway. We report that PRL induces rapid increases in two novel inositol phospholipids, almost certainly PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Pre-traitment of CHO cells with wortmanin, a specific PtdIns3-kinase inhibitor, considerably reduces the PRL-induced increase in PtdInd(3,4,5)P3, thus suggesting an involvement of this enzyme in the cascade of activation of cytoplasmic kinase proteins. A pathway beginning with the activation of PtdIns3-kinase, phosphorylation of PtdIns(4,5)P2 and rapid synthesis of PtdIns(3,4,5)P3 is proposed. PtIns(3,4,5)P3 may acts as a lipid second messenger, directly or indirectly responsible for some of the multiple cell changes attributed to PRL.