Abstract
Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, β-PMA or β-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km . Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, β-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetineKd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.
Footnotes
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Send reprint requests to: Dr. Randy D. Blakely, Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-6600.
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↵1 This work was supported by NINDS Award NS33373 and MH58921 to R.D.B.
- Abbreviations:
- DA
- dopamine
- NE
- norepinephrine
- NET
- norepinephrine transporters
- PKC
- protein kinase C
- hNET
- human NET
- mAChR
- muscarinic M3 acetylcholine receptor
- KRH
- Krebs-Ringers-HEPES
- PBS
- phosphate-buffer saline
- TBS
- Tris-buffered saline
- NDS
- normal donkey serum
- PDBu
- phorbol-12,13-dibutyrate
- PMA
- phorbol-12-myristate-13-acetate
- SDS
- sodium dioctyl sulfate
- U-0521
- 3,4-dihydroxymethyl propiophenone
- PMSF
- phenylmethylsulfonyl fluoride
- Received January 13, 1998.
- Accepted June 8, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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