Technical Reference #1693

1693.

Passive Diffusion of Naltrexone Into Human and Animal cells and Upregulation of Cell Proliferation Fan Cheng; Patricia J. McLaughlin; William A. Banks and Ian S. Zagon;, The Pennsylvania State University, American Journal of Physiology: Regulatory; Integrative and Comparative Physiology, 76(1693), (2009)
Link To Paper

Abstract:
Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells NTX was fluorescently labeled (FNTX [1-(N)-fluoresceinyl naltrexone thiosemicarbazone) to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line SCC-1 was incubated with FNTX for as little as 1 min cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopywith enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2) African green monkey kidney cell line COS-7 and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 40C with signal being comparable to that recorded at 370C . A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis cells incubated with FNTX at concentrations ranging from 10-5 to 10-8 M had a BrdU index that was 39-82% greater than for vehicle treated cells and was comparable to that of unlabeled NTX (37-70%). Taken together these results suggested that NTX enters cells by passive diffusion in a non-saturable manner.

Keywords:
Naltrexone - Transport - Diffusion - Tissue Culture - Cell Proliferation

Materials & Methods:
FNTX uptake studies. For experiments on FNTX uptake SCC-1 MIA PaCa-2 COS-7 and hMSCs cells were grown to 50-80% confluence in 35-mm glass bottom culture 6 dishes (MatTek Ashland MA) prior to incubation with FNTX or other reagents. FNTX was added to log phase cell cultures for 1 5 15 30 or 60 min at 370C. FNTX was aspirated incubated in warm media and immediately analyzed by epi-fluorescent phase or differential contrast interference (DIC) microscopy with an IX-81 Olympus Inverted microscope (Center Valley PA). Some images were deconvolved using the constrained iterative algorithm in Slide Book (Intelligent Imaging Innovations Denver CO) at 40X objective. For each image Z stacks of 12 images were obtained with each image taken 0.5 μm apart. In addition these cell cultures were analyzed by microscopy at 24 48 and 72 h following removal of FNTX.

Microscopic Technique
differential contrast interference (DIC) microscopy

Cell Type(s)
SCC-1