Abstract

We have used human erythroleukemia (HEL) cells to investigate distal signaling mechanisms of neuropeptide-Y (NPY) receptors. NPY did not activate phospholipase D, determined as a phosphatidylethanol formation, or protein kinase C (PKC) determined enzymatically as a translocation to the plasma membrane. However, NPY caused a rapid (already maximal after 30 s) and concentration-dependent (maximum at 10–100 nM) activation of extracellular signal-regulated kinase (ERK) as assessed by immunoblotting with epitope-specific, antiphosphotyrosine antibodies and in some cases enzymatically. ERK activation by 100 nM NPY was abolished by the Y₁ NPY receptor antagonist BIBP 3226 (1 μM), pertussis toxin treatment (100 ng ml⁻¹ overnight), the mitogen-activated protein kinase (MAPK) kinase inhibitor PD 98059 (100 μM), and the phosphatidylinositol-3-kinase inhibitor wortmannin (100 nM). Whereas the PKC inhibitor staurosporine (3 μM) inhibited ERK activation by NPY, the chemically distinct PKC inhibitors calphostin C (3 μM), Gö 6976 (3 μM), and bisindolylmaleimide I (3 μM) did not. NPY did not activate other MAPK such as jun N-terminal kinase or p38 MAPK. We conclude that NPY does not activate phospholipase D, PKC, jun N-terminal kinase, or p38 MAPK in HEL cells. However, NPY activates ERK by a pathway involving Y₁ receptors, pertussis toxin-sensitive G proteins, and phosphatidylinositol-3-kinase, whereas PKC may not be involved. Staurosporine may have PKC-independent effects on ERK activation.