Imidazoline receptor proteins are regulated in platelet-precursor MEG-01 cells by agonists and antagonists

The I1-imidazoline receptor is a novel brainstem modulator of sympathetic outflow that is elevated on platelets and in brains of depressed patients. A positive correlation has been reported (accompanying manuscript) between plasma norepinephrine (NE) concentrations and the densities (Bmax) of platelet I1 binding sites (I1 sites). I1-candidate proteins of 33 kDa and 85 kDa are now identified on Western blots probed with anti-imidazoline receptor antiserum (IRBP antiserum), that correlate with Bmax values for I1 sites. Furthermore, a human megakaryoblastoma cell line (MEG-01) has been used to study the regulation of these proteins on megakaryocytic cells, while bovine adrenal chromaffin cells provide a standard I1 cell type for comparison. Both the 33 kDa and 85 kDa IRBP-immunoreactive bands were enriched in plasma membrane fractions. IRBP antiserum did not cross-react with I2 imidazoline binding sites located on platelet mitochondrial membranes. The 85 kDa band was enhanced under conditions lacking fetal bovine serum (FBS) from the culture medium 6 h prior to harvesting. Conversely, 33 kDa protein was enhanced on MEG-01 cells grown in the presence of 10% FBS; suggesting that a precursor (85 kDa) and product (33 kDa) relationship might be induced by serum. The 85 kDa band was robustly up-regulated in response to imidazoline receptor-sensitive ligands; moxonidine, idazoxan and agmatine (10 microM each for 6 h). NE also up-regulated the 85 kDa IRBP-immunoreactive protein on MEG-01 membranes, but to a lesser extent. Idazoxan, an imidazoline alpha 2-antagonist, off-set its induction of 85 kDa protein by reducing the 33 kDa band. Yohimbine, a non-imidazoline alpha 2-antagonist, was ineffective alone, or in combination with moxonidine (up to 40 microM), but yohimbine blocked NE's induction of the 85 kDa band. Therefore, a rise in either plasma NE and/or endogenous I-site ligands (i.e. agmatine) could explain an elevation of imidazoline receptors observed in depression.