Kelly, E. | bioRxiv Science

bioRxiv2019

Fentanyl depression of respiration: comparison with heroin and morphine

Background and PurposeFentanyl overdose deaths have reached epidemic levels in North America. Death in opioid overdose invariably results from respiratory depression. In the present work we have characterized how fentanyl depresses respiration and by comparing fentanyl with heroin and morphine, the active breakdown product of heroin, we have sought to determine whether there are factors, in addition to high potency, that contribute to the lethality of fentanyl.\n\nExperimental ApproachRespiration (rate and tidal volume) was measured in awake, freely moving mice by whole body plethysmography\n\nKey ResultsIntravenously administered fentanyl produced more rapid depression of respiration than equipotent doses of heroin or morphine. Fentanyl depressed both respiratory rate and tidal volume, the effect on tidal volume may reflect increased respiratory muscle stiffness. Fentanyl did not depress respiration in opioid receptor knock-out mice. Naloxone, the opioid antagonist widely used to treat opioid overdose, reversed the depression of respiration by morphine more readily than that by fentanyl whereas diprenorphine, a more lipophilic antagonist, was equipotent in reversing fentanyl and morphine depression of respiration. Prolonged treatment with morphine induced tolerance to respiratory depression but the degree of cross tolerance to fentanyl was less than the tolerance to morphine itself.\n\nConclusion and ImplicationsWe propose that several factors (potency, rate of onset, muscle stiffness, lowered sensitivity to naloxone and lowered cross tolerance to morphine) combine to make fentanyl more likely to cause opioid overdose deaths than other commonly abused opioids.

pharmacology and toxicology↗

bioRxiv2019

Cell-projection pumping: A hydrodynamic cell-stiffness dependent mechanism for cytoplasmic transfer between mammalian cells

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fib) and malignant cells (MC). Others report similar transfer via either tunneling nanotubes (TNT) or shed membrane vesicles and this changes the phenotype of recipient cells. Our current time-lapse microscopy showed most exchange was from Fib into MC, with less in the reverse direction. Although TNT were seen, we were surprised transfer was not via TNT, but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless, by their organellar cargo and the grooves they formed indenting MC, while this was consistent with holotomography. Discrete, rapid and highly localized transfer events, evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell-projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell-projections normally returns cytoplasm to the cell body. We hypothesize cell-projection pumping (CPP), where cytoplasm in retracting cell-projections partially equilibrates into adjacent recipient cells via micro-fusions that form temporary inter-cellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modelling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure towards least resistance. Predictions from the model were satisfied when Fib were co-cultured with MC, and fluorescence exchange related with cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MC or Fib was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian inter-cellular cytoplasmic transfer and communication. SIGNIFICANCETime-lapse observations of co-cultured cells led us to hypothesize what we believe to be a novel hydrodynamic mechanism transferring cytoplasm between cells. Similar transfer by other mechanisms markedly affects cell behavior. Combined mathematical modelling, satisfaction of predictions from the mathematical model in cell culture experiments, and separate computer simulations that generate outcomes similar to experimental observations, support our hypothesized mechanism.

cell biology↗