Long-term hypoxia (LTH) induces profound changes in vascular reactivity in both fetal and adult cerebral blood vessels. Although endothelial cells lining the blood vessel lumen and peripheral adrenergic nerves play major roles in vessel reactivity, the vascular smooth muscle cells (VSMCs) are the final arbiters of vascular tone. VSMCs normally exist in a partially contracted state, giving rise to myogenic tone, and are able to contract further or to relax upon exposure to soluble signals. VSMC responsiveness is influenced by many factors, including development and exposure to long-term hypoxia (LTH), both of which are principal variables in the proposed studies. Developmental changes in VSMCs include changes in contractile proteins, membrane composition, receptors, signal pathways, and ion channels. Each of these changes influences vascular reactivity. Current research from our laboratory indicates that some of the effects of LTH on vascular smooth muscle involve calcium-activated, voltage-sensitive K+ (BK or KCa) channels. BK channels are arguably the single most important channels on VSMCs of the cerebral and pulmonary branches of the vascular tree. BK channels are pivotal in terms of VSMC reactivity because they couple free intracellular calcium levels to membrane potential. In addition, they are modulated by more extra- and intracellular factors than any other known VSMC channels. Indeed, BK channels are found in all types of smooth muscle where their activation decreases contractility indirectly by hyperpolarizing the membrane. In vascular smooth muscle from mature arteries, BK channels regulate membrane potential by responding to local elevations of [Ca2+]i from """"""""Ca 2+ sparks"""""""" released from internal stores under the control of ryanodine receptors. However, in fetal cerebral VSMCs, BK channels respond primarily to global elevations of [Ca2+]i due to Ca 2+ influxing through L-type calcium channels. BK channel function and structure can vary due to alternative splicing of a single SIo gene, but little is known about channel functional and structural changes during development or LTH. The BK channels of VSMCs and brain neurons associate functionally in the plasma membrane with certain protein kinases and phosphatases. We find that the responses of BK channels to various modulatory factors differ both during development and in response to LTH, indicating that the physiological adaptations of BK channels to LTH differ between the fetus and adult, some of which are potentially critical to pathophysiological survival. These include the following: (1) In LTH adults the Ca 2+ set point of BK channels (3.3 mu M) is similar to that of the LTH and normoxic fetus (3.0 and 4.7 mu M, respectively), but half that of normoxic adults (8.8 mu M; Lin et al., 2003). (2) In LTH adults the sensitivity of BK channels to inhibition by intracellular hemin (IC50 = 50 nM) is four times that of the normoxic adult or the LTH and normoxic fetus (IC50 = 200 nM). (3) In LTH adults the BK channels are activated by acute hypoxia to more than twice that of BK channels in the LTH fetus. (4) In LTH adults BK channels have almost twice the channel-associated protein kinase A (PKA) activity as that of the LTH fetus, but, unlike that of the LTH fetus, exhibit virtually no associated protein kinase G (PKG) activity. We also find identical actions of exogenous kinases and phosphatases on voltage activation of BK channels from fetal and adult ovine basilar arteries. This suggests that the same channel splice variant is expressed throughout development. However, our findings also indicate that these enzymes produce dissimilar actions on BK channels from LTH versus normoxic animals, regardless of developmental stage. This suggests that different splice variants occur under LTH and normoxia. In our proposed study we have opted to use both pulmonary, as well as cerebral, arteries because pulmonary vessels react oppositely to cerebral arteries in response to acute hypoxia, and nothing is yet known concerning their responses to LTH, the differences associated with developmental age, or their sensitivity to hemin.
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