Ca²⁺signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Abstract

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.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth

“…Images were acquired at a 12-bit sampling depth with use of a water-immersion ϫ40 Plan-Apochromat, 1.0 numerical aperture objective mounted on an InverterScope objective inverter (LSM Tech, Wellsville, PA), which allowed for imaging in an upright configuration. To prevent arterial movement during recordings, arteries were pretreated for 1 h with a cocktail including 10 M Y27632 to inhibit Rho kinase, 10 M cytochalasin D to inhibit actin polymerization, and 10 M W-7 to inhibit myosin light chain kinase (10,43,56,88). The concentrations of these drugs were chosen on the basis of the outcomes of wire myography studies where the influence of the drugs on vessel reactivity were examined individually.…”

Combination Therapy With Fasudil and Sildenafil Ameliorates Monocrotaline-Induced Pulmonary Hypertension and Survival in Rats

“…Thus, fasudil blocks vasoconstriction through the dephosporylation of myosin phosinduce vasodilation especially where vasoconstrictor tone is increased by a variety of mechanisms including hypoxia, NOS inhibition and other mechanisms. 37 In contrast, sildenafil inhibits pulmonary vascular remodeling by a different mechanism targeting the eNOS-NO-cGMP pathway. 38 Thus, Rho-kinase inhibition and PDE5 inhibition might have additive and synergistic effects in terms of vascular dilatation and remodeling (Figure 7).…”

Hypoxia. 4. Hypoxia and ion channel function

“…Some NSCCs are highly Ca 2ϩ permeable; both store-operated and receptor-operated Ca 2ϩ channels, which are likely composed of canonical transient receptor potential (TRPC) proteins, fall into this category and are expressed in PASMCs (135,136,236). Acute hypoxia increased Ca 2ϩ influx through store-operated Ca 2ϩ channels, which may be due to Ca 2ϩ release from the SR (107) or phosphorylation of the channels by Rho kinase or myosin light chain kinase (238). In addition to being Ca 2ϩ permeable, these channels can also conduct Na ϩ influx, allowing for the possibility that opening of these channels also contributes to depolarization and activation of VGCC.…”

“…Instead, inhibitors of NSCCs reduced both [Ca 2ϩ ] i and tone in pulmonary vascular smooth muscle from chronically hypoxic rats (127,239). NSCCs are not activated by depolarization, but their activity may be influenced by phosphorylation (93,156,238). Increased abundance of TRPC protein was observed in pulmonary arterial smooth muscle from chronically hypoxic animals (127,239), and in PASMCs exposed to prolonged hypoxia in vitro, through a HIF-1-dependent mechanism (239), leading to a sustained elevation in [Ca 2ϩ ] i .…”