Histamine H1 and H3 receptor activation increases the expression of Glucose Transporter 1 (GLUT-1) in rat cerebro-cortical astrocytes in primary culture.

Astrocytes take up glucose via the 45 kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1 h incubation) significantly increased GLUT-1 mRNA to 153 ±â€¯7, 163 ±â€¯2 and 168 ±â€¯13% of control values, respectively. In immunoblot assays, incubation (3 h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224 ±â€¯12, 305 ±â€¯11 and 193 ±â€¯13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) → diacylglycerol (DAG)/Ca2+→ PKC and PLC → DAG/Ca2+ → PKC → MAPK pathways.

Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 µM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.