Hepatocyte growth factor-induced enhancement of dendritic branching is blocked by inhibitors of N-methyl-D-aspartate receptors and calcium/calmodulin-dependent kinases.
Hepatocyte growth factor (HGF) and its receptor, Met, are clustered at excitatory synapses and can enhance N-methyl-D-aspartate (NMDA) receptor current and promote formation of neurites and dendrites. In this study, we examine the effects of HGF on dendritic arborization in mature cultures of dissociated hippocampal neurons. Exogenous HGF treatment caused a dose-dependent increase in total dendritic branch tip number, total dendritic branch length, and dendritic complexity in these neurons. NMDA receptor activity has been linked to changes in dendritic structure, so we tested the effects of HGF on the dendritic arbor in the presence of DL-2-amino-5-phosphonopentanoic acid (APV), an NMDA receptor inhibitor. APV blocked the HGF-induced enhancement of the dendritic arbor in a dose-dependent manner. Similarly, pretreatment of neurons with KN62, an inhibitor of calcium-dependent kinases, suppressed changes in dendritic branching induced by HGF. These results suggest that HGF initiates Ca2+-dependent processes, so we examined the effect of HGF on intracellular calcium levels and autophosphorylation of the calcium/calmodulin-dependent protein kinase II (CaMKII). HGF caused a persistent increase in fluorescence in clusters along dendrites of neurons preloaded with the Ca2+ indicator Fluo-4. HGF treatment also enhanced autophosphorylation of CaMKII. The increases in Fluo-4 fluorescence and autophosphorylation of CaMKII were blocked by pretreatment of neurons with APV. These results indicate that HGF stimulates Ca2+ influx into dendrites through the NMDA receptor and that this effect is necessary for the changes in dendritic morphology induced by HGF.
Tyndall
Stephanie J
SJ
Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USA.
Patel
Sandip J
SJ
Walikonis
Randall S
RS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; C063302:KN 62; D017228:Hepatocyte Growth Factor; C056639:2-amino-5-phosphopentanoic acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D014633:Valine; D002118:Calcium