C - Michigan Technological University

are also 5HT-1A, -2A, and -1D receptors and 5HTT transporters on osteoblasts [270],none of which would have been blocked by SB206553; therefore, serotonin has notbeen definitely ruled out as a potential apoptosis effector in this system.H-89 inhibits the PKA pathway, an internal signaling cascade which, inosteoblasts, responds to a variety of hormones including parathyroid hormone, calcitoningene-related peptide, and norepinephrine [271-274]. H-89 can suppress anti-apoptoticsignaling in osteoblasts [272, 275, 276]; however, H-89 does not significantly suppressthe seasonal difference in apoptotic response of pre-osteoblasts primed in bear serum(Figure 5.5), suggesting that the PKA pathway is not vital to this seasonal difference incaspase-3/7 response. Pertussis toxin inhibits the g-protein subunit G i . A variety ofhormones and factors can function through the G i subunit of g-protein coupled receptors,including parathyroid hormone, aluminum, lysophosphatidic acid, epinephrine, andmelatonin [277-280]. Pertussis toxin can suppress anti-apoptotic signaling in osteoblasts[281, 282]. Pertussis toxin did not greatly alter the seasonal difference observedbetween pre-osteoblasts primed in prehibernation and hibernation serum samples(Figure 5.6), suggesting that the G i subunit is not necessary for this seasonal differencein caspase-3/7 response.In an attempt to determine the size of the serum factor responsible for theseasonal difference in apoptotic response, molecules larger than 10 kD were filtered outof the media before stimulation. Samples were treated with filtered and unfiltered serumfor 24 hours, followed by a 6 hour serum starvation, as in previous experiments. Therewas not a seasonal difference between cells primed in filtered prehibernation andhibernation serum samples (Figure 5.7 B); however, there was also no differencebetween starved and unstarved cells which were primed in filtered 2% FBS (Figure 5.7A), suggesting that the 24 hour priming in filtered media suppresses caspase-3/7response at this time-point. The caspase-3/7 response to starvation is time-dependent(Figure 5.1), and we thought that the filtered media might induce a starvation-like statein the cells. Therefore, we ran a second experiment in which cells were cultured infiltered 2% FBS or serum free media for 6 hours, followed by caspase-3/7 activityanalysis. In this second experiment, there was a significant difference between the 2%FBS and the serum-free media (Figure 5.8 A). We then cultured cells for 6 hours in80