These data are consistent with leptin activation of KATP channel currents involving a phosphorylation-dependent process

These data are consistent with leptin activation of KATP channel currents involving a phosphorylation-dependent process. H-7 does not occlude leptin activation of KATP channels Since protein kinases are capable of phosphorylating serine/threonine residues of a range of proteins we examined their role in the actions of leptin using the broad-spectrum protein kinase inhibitor H-7 (Hidaka, Watanabe & Kobayashi, 1991). or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 M) prevented the actions of both leptin and tyrphostin B42. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42. It is well established that protein tyrosine kinases regulate a variety of cellular functions including proliferation, differentiation and signalling processes. Although a number of distinct tyrosine kinases and phosphatases Epimedin A1 have been identified (Levitzki & Gazit, 1995), the physiological actions and the intracellular targets of these proteins remain unclear. There is, however, increasing evidence that tyrosine kinases and phosphatases can modulate a variety of ion channels by either increasing or decreasing channel activity (Siegelbaum, 1994). In pancreatic -cells (Keiffer, Heller, Leech, Holz & Habener, 1997) and the CRI-G1 insulin-secreting cell line (Harvey, McKenna, Herson, Spanswick & Ashford, 1997), leptin, the gene product, activates ATP-sensitive potassium (KATP) channels, an action consistent with suppression of insulin secretion. The leptin receptor shows sequence homology with members Mouse monoclonal to KID of the class I cytokine receptor superfamily (Tartaglia 1995), which are thought to signal via association with tyrosine kinases of the janus kinase (JAK) family. Indeed, the long form of the leptin receptor (OB-Rb) activates JAK2 in a haematopoetic cell line (Ghilardi & Skoda, 1997). Several pathways can be activated by JAKs including the insulin receptor substrate proteins (Ihle, 1995). Phosphoinositide 3-kinase (PI 3-kinase) is just one of many proteins associated with the signalling downstream of insulin receptor substrate-1 (IRS-1; Myers & White, 1996). Recently, we have shown that the ability of leptin to activate KATP channels is not only regulated by insulin but also that the pathway underlying this action of leptin involves activation of PI 3-kinase (J. Harvey & M. L. J. Ashford, unpublished observations). Prolactin is also capable of activating JAK2 (Prevarskaya, Skryma, Vacher, Daniel, Djiane & Dufy, 1995) and PI 3-kinase (Berlanga, Gualillo, Buteau, Applanat, Kelly & Edery, 1997), thus the signalling capabilities of the leptin receptor in CRI-G1 cells may show parallels to those of other class I cytokine receptors. Since tyrosine phosphorylation plays a critical role in the actions of other cytokines, we have examined the effects of inhibitors of tyrosine kinases and phosphatases Epimedin A1 in the present study, in order to elucidate further the mechanism underlying leptin activation of KATP channels in CRI-G1 insulinoma cells. In addition to protein tyrosine kinases, the activity of ion channels can be modulated by serine/threonine-specific protein kinases (Jonas Epimedin A1 & Kaczmarek, 1996). Epimedin A1 Indeed phorbol ester-induced activation of protein kinase C results in phosphorylation and subsequent activation of KATP channels in insulin-secreting cells (Ribalet, Eddlestone & Ciani, 1988; De Weille, Schmid-Antomarchi, Fosset & Lazdunski, 1989). Furthermore, in another insulin-secreting cell line RINm5F (Ribalet, Ciani & Eddlestone, 1989) and rabbit arterial smooth muscle (Quayle, Bonev, Brayden & Nelson, 1994), KATP channel activity is enhanced via protein kinase A-dependent phosphorylation. Consequently we have also examined whether leptin activates KATP channels in CRI-G1 cells via serine/threonine-specific protein kinases. We have reported previously that tyrosine kinase inhibitors mimic leptin activation of KATP channels in CRI-G1 insulin-secreting cells (Ashford & Harvey, 1997). METHODS Cell culture Cells from the rat insulin-secreting cell line CRI-G1 were grown in Dulbecco’s modified Eagle’s medium with sodium pyruvate and glucose (Life Technologies), supplemented with 10 %10 % fetal calf serum (Sigma) and.