| Abstract No.: | A-E1168 |
| Country: | Canada |
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| Title: | PKG ACTIVATION INCREASES SENSITIVITY OF MOUSE RESPIRATORY RHYTHM GENERATION TO HYPOXIC AND HYPERTHERMIC STRESS. |
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| Authors/Affiliations: | 1 Gary A.B. Armstrong*, 2 Juan Lopez-Guerrero, 3 Ken Dawson-Scully, 2 Fernando Pena, 1 Meldrum Robertson, 1 Queen's University, Department of Biology, Kingston, ON, Canada, 2 Cinvestav-IPN, Departamento de Farmacobiologia, Mexico, 3 University of Toronto, Department of Biology, Mississauga, ON, Canada |
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| Content: | Objectives: At elevated body temperatures respiratory rhythm At elevated body temperatures, respiratory rhythm generation can become impaired. If body temperatures continue to rise unabated, fever-induced respiratory arrest and death can occur. Pharmacological inhibition of the protein kinase G (PKG) pathway in insects confers neuronal tolerance to heat stress. We examined the role PKG inhibition may have on neuronal circuit protection against thermal and hypoxic stress of the mammalian medullary brainstem slice containing the respiratory rhythm generating circuit (Pre-Bötzinger complex (PBC)).
Materials and Methods: Rhythmic brainstem slices were obtained from 5 or 6 day old mice and bathed in artificial cerebrospinal fluid (ACSF), aerated with carbogen (95% O2 and 5% CO2) and held at 30°C. Suction electrodes were placed near the PBC to monitor fictive respiratory activity. ACSF temperature was gradually raised in a ramp-like manner (5°C/min) until rhythm failure occurred thereupon the temperature was allowed to return to 30°C allowing for spontaneous rhythm generation to recover. The temperature at which rhythm generation failed and the length of time taken to recover were measured. A second hyperthermic challenge was performed 20 minutes following rhythm recovery, however, after 10 minutes, ACSF containing 10 µM 8-Br-cGMP (PKG agonist) was used to bathe the slice. Slices designated as controls were continuously bathed in ACSF. In a second set of experiments rhythmic slices were placed under hypoxic conditions (carbogen replaced with nitrogen) for 10 minutes. Changes in rhythm frequency, irregularity of the rhythm and the rise time of the integrated fictive respiratory activity trace were quantified following pharmacological manipulation of the PKG signaling pathway.
Results: At 30°C fictive rhythm frequency was 0.25 ± 0.04 Hz. Frequency of fictive respiratory rhythm did not change following bath application of 8-Br-cGMP. During the temperature ramp frequency increased to 0.62 ± 0.04 Hz just prior to failure in control slices and 0.59 ± 0.05 Hz in 8-Br-cGMP-treated slices. At 30°C control slices had a rhythm irregularity score of 0.17 ± 0.01 whereas slices treated with 8-Br-cGMP were much more irregular (0.43 ± 0.07). During hyperthermia, slices treated with 8-Br-cGMP failed at lower temperatures than controls. Furthermore, the length of time required to restore spontaneous rhythm generation was markedly longer in slices treated with 8-Br-cGMP. During hypoxia, fictive inspiratory burst activity usually changes from an augmenting to a decrementing bursting pattern (gasping). Slices treated with 8-Br-cGMP did not change to a decrementing bursting pattern.
Conclusion: Signaling pathways regulate numerous cellular processes including the physiological adaptations that switch on and provide neuroprotection during stress. We have demonstrated that activation of the PKG signalling pathway impairs the rhythmic medullary brainstem slice’s ability to withstand thermal and hypoxic stresses. We are currently testing if inhibition of this signalling pathway confers protection against thermal stress. Here we show that PKG activation modulates mammalian respiratory rhythm generation and impairs this circuit’s tolerance to heat stress and hypoxia.
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