(2,3,7,8,38,41,45,47,58,59,70) and contraction (20,24,26,28,32 ] i responses to hypoxia in isolated PA (43). One possible explanation for these findings is that hypoxia caused release of Ca 2ϩ from SR in PASMC, leading to SR depletion, activation of store-operated Ca 2ϩ channels (SOCC), and capacitative Ca 2ϩ entry (CCE). Recently, we reported that several so-called "canonical transient receptor potential (TRPC)" proteins were expressed in smooth muscle of distal PA (60), which are thought to be the major vascular locus of HPV (49). These proteins are homologs of TRP and TRP-like proteins that make up Ca 2ϩ channels in Drosophila photoreceptors and are thought to compose mammalian SOCC, many forms of which may also be permeable to Na ϩ and other cations and therefore function as nonselective cation channels (NSCC) (34, 37, 51). Consistent with TRPC expression, we and others demonstrated the presence of CCE in distal PA (51, 60). More recently, we found that acute hypoxia increased [Ca 2ϩ ] i , Ca 2ϩ influx, and CCE in distal PASMC and that these effects were completely blocked by removal of extracellular Ca 2ϩ or SOCC/NSCC antagonists, but not nifedipine (61). These findings are consistent with recent observations from other laboratories (21,22,35) and suggest that HPV may require activation of SOCC in PASMC; however, it is well known that cell isolation and culture can alter cell phenotype. Moreover, changes in PASMC [Ca 2ϩ ] i do not necessarily translate to changes in pulmonary vascular resistance. In the present study, therefore, we assessed the contribution of SOCC and CCE to HPV in isolated lungs, where physiologically relevant pulmonary vasomotor responses can be measured directly. METHODS Isolated Lung PreparationOur protocol was approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University. Male Wistar rats (200 -400 g) were anesthetized with pentobarbital sodium (65 mg/kg ip). A tracheostomy was performed, and the animal was ventilated with room air at a tidal volume of 10 ml/kg and a rate of 30 min Ϫ1(Harvard Rodent Ventilator model 883; Harvard Apparatus, Holliston, MA). A thoracotomy was performed, heparin (100 units) was injected into the right ventricular cavity, and the animal was exsanguinated from the femoral artery. The ventilating gas was changed to 16% O 2-5% CO2. Cannulae were inserted into the main PA and left atrium, which drained into a heated reservoir. The lungs were perfused with
Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca(2+)](i). To determine whether MLCK and ROK antagonists alter Ca(2+) signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca(2+)](i), Ca(2+) entry, and Ca(2+) release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca(2+)](i) in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca(2+) release from the sarcoplasmic reticulum and Ca(2+) entry through store- and voltage-operated Ca(2+) channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca(2+) signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca(2+) signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.
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