| Previous research from our laboratory has shown that myometrial contractility is reduced in pregnant sheep exposed to long-term hypoxia (LTH). The studies in this dissertation examined the effect of LTH on various aspects of Ca2+ regulation, large-conductance, Ca2+-activated (BK) channels activity and protein expression, and cGMP-dependent protein kinase (PKG) activity. Myometrium was collected from near-term pregnant sheep exposed to high altitude-associated LTH and from normoxic pregnant controls. After pretreatment with 10-8 M oxytocin (OT), circular and longitudinal myometrial strips were treated with nifedipine (L-type Ca 2+ channel blocker), ruthenium red, ryanodine (blockers of IP 3-sensitive stores), 2-Nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; phospholipase C inhibitor), tetraethylammonium (TEA; nonspecific K + channel inhibitor), iberiotoxin (IbTX; specific blocker of BK channels), or KT-5823 (PKG inhibitor). Western blots were also conducted for BKalpha and BKbeta subunit protein. LTH increased the maximum relaxation response to nifedipine whereas no significant differences were observed in response to ruthenium red or ryanodine. The maximum relaxation response to NCDC was lower in the LTH circular layer. No significant differences in response to TEA were found; however, the maximum tension response to IbTX in the LTH group was significantly increased. Western blot analysis revealed two beta1 subunit bands (38 and 48 kDa), but did not show any significant differences between treatment groups or layers. However, the BK beta:alpha subunit ratio (using either the 48 kDa beta band only or combined with the 38 kDa beta band) showed a significant decrease in the circular layer after exposure to LTH. After treatment with KT-5823, the contractility of LTH tissue significantly decreased in both layers. We conclude that LTH decreases reliance on IP 3-associated Ca2+ stores and increases dependence upon Ca2+ entry through L-type Ca2+ channels. The contribution of SR Ca2+ is negligible. In addition, BK channel activity is enhanced following LTH, possibly mediated by a decreased beta:alpha subunit ratio. Finally, PKG inhibitory activity may be suppressed in LTH myometrium with a possible switch to stimulatory in the circular muscle. |
