PROLACTIN INDUCES AN INWARD CURRENT THROUGH VOLTAGE-INDEPENDENT CA2-HAMSTER OVARY CELLS STABLY EXPRESSING PROLACTIN RECEPTOR( CHANNELS IN CHINESE)

There is still only limited understanding of the early steps of prolac tin (PRL) signal transduction in target cells. Recent studies have ide ntified some of the essential first steps: these include the rapid ass ociation of the PRL receptor with JAK tyrosine kinases and tyrosine ph osphorylation of a number of proteins, including members of the signal transducer and activator of transcription (Stats) family. On the othe r hand, binding of PRL to its receptor is rapidly followed by calcium influx. However, PRL-induced ionic events and the related ionic channe ls involved have not been clearly established. This work was undertake n to characterise tbe channels responsible for calcium influx and to o btain an insight into their activation processes. Using the patch-clam p technique in the cell-attached configuration, single Ca2+ channel cu rrents were recorded following PRL application (10 nM) in Chinese hams ter ovary (CHO) cells stably expressing PRL receptor (CHO-E32). Statis tical analysis showed that the recorded currents were voltage-independ ent, with a slope conductance of 16 pS. Although these channels were p resent in excised patches, the fact that PRL was unable to activate th em suggested that a soluble cytoplasmic component may be required. App lication of the purified inositol phosphate, Ins(1,3,4,5)P4 (2 mu M), to the inside of the excised patch membrane activated the voltage-inde pendent 16 pS Ca2+ channel. The open probability (Popen) was enhanced. The inositol phosphates Ins(1,2,3,3,5)P5 and Ins(1,4,5)P3 did not aff ect channel activity while InsP6 (20 mu M) had some effect, although l ess 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 30s). Conversely, Ins( 1,4,5)P3 showed a transient decrease at 5 s, accompanied by a concomit ant increase in Ins(1,3,1,5)P4, suggesting that the former could be tr ansiently phosphorylated to produce the latter. Comparison of the prod uction kinetics of Ins(1,4,5)P3, Ins(1,3,4,5)PI, InsP5, and InsP6 indi cated the possibility of additional metabolic routes which have yet to be determined. This study suggests that PRL promotes Ca2(+) entry thr ough voltage-independent Ca2+ channels that may be activated by Ins(1, 3,4,5)P4 and InsP6.