Related Experiment Videos
Early alterations of actin cytoskeleton in OK cells by opioids
E A Papakonstanti1, E Bakogeorgou, E Castanas
1Department of Biochemistry, University of Crete, School of Medicine, Heraklion, Greece.
Abstract:
Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids alphaS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and alphaS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells.
Insights
Opioids rapidly alter actin cytoskeleton dynamics in OK cells, affecting actin polymerization and microfilament organization. These opioid-induced changes may explain their antiproliferative effects on cell growth.
Area of Science:
- Cell Biology
- Pharmacology
- Biochemistry
Background:
- Opioid binding sites have been identified in OK (opossum kidney) cells.
- Opioids have been observed to decrease the proliferation of these cells.
Purpose of the Study:
- To investigate the effects of opioids on the actin cytoskeleton in OK cells.
- To determine if opioid-induced antiproliferative actions are related to alterations in actin microfilaments.
Main Methods:
- Exposure of OK cells to specific opioids (alphaS1 casomorphin, ethylketocyclazocine).
- Quantitative fluorescence, DNase I inhibition assay, and confocal laser scanning microscopy to assess actin dynamics and organization.
- Northern blot analysis for actin transcript levels and use of opioid antagonist (diprenorphine) and microfilament stabilizer (phallacidin).
Main Results:
- Opioids caused rapid, dose-dependent reorganization of actin microfilaments, decreasing F-actin and increasing the G/total-actin ratio.
- Opioid effects on actin polymerization were reversible and did not affect microtubules or vimentin.
- Actin transcript levels remained unchanged, and diprenorphine blocked opioid effects.
Conclusions:
- Opioids, acting via kappa 1 binding sites, rapidly modify actin polymerization dynamics and microfilament organization in OK cells.
- These opioid-induced cytoskeletal changes are linked to their antiproliferative effects on cell proliferation.