Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia

Abstract

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 μM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-β-cyclodextrin (mβCD; 10 mM). Cholesterol depletion with mβCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mβCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 μM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.

Pathogenesis of pulmonary hypertension: a case for caveolin-1 and cell membrane integrity

“…Chronic hypoxia leads to a reduction in the membrane cholesterol and impairs agonist-induced Ca 2ϩ entry. The introduction of cell permeable CSD restores Ca 2ϩ entry in these cells (97). These observations indicate that caveolin-1 may be required for Ca 2ϩ entry in ECs for vasoactivity.…”

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…We, therefore, postulated that TRPV4 channels in ECs from small PAs couple to novel physiological activators. Previous studies in large PAs revealed that endogenous purinergic receptor activator ATP increases endothelial Ca 2+ and causes endothelium‐dependent vasorelaxation 22, 23, 24, 46, 47. We, therefore, hypothesized that ATP dilates small PAs via activation of TRPV4 sparklets.…”

“…In this regard, purinergic receptor agonist ATP has been shown to increase endothelial Ca 2+ and cause vasodilation in large, conduit PAs. [22][23][24] ATP can be released into the circulation by ECs, smooth muscle cells (SMCs), and red blood cells. 25,26 Circulatory ATP may, therefore, serve as an important regulator of pulmonary vascular resistance.…”

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection

“…In coronary and other vascular smooth muscles, M␤CD treatment modifies ion channel function (BK Ca and Ca 2ϩ -activated Cl Ϫ currents) and receptordependent control of intracellular Ca 2ϩ concentration and vascular tone (40,47,51). M␤CD also modifies BK Ca currents in nonvascular smooth muscle (50), together with BK Ca activity (43) and agonist-mediated Ca 2ϩ entry (30) in the vascular endothelium. Interestingly, M␤CD treatment diminishes storeoperated Ca 2ϩ channel function (where the reduction in intracellular Ca 2ϩ stores activates Ca 2ϩ influx) and attenuates the hypertensive phenotype (associated with elevated caveolae/ caveolin levels) in pulmonary arteries from patients with idiopathic pulmonary arterial hypertension (33).…”

“…In other vascular smooth muscles, M␤CD enhances Ca 2ϩ -activated Cl Ϫ currents (51), and in uterine myocytes, M␤CD amplifies large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channel currents (50). In the vascular endothelium, M␤CD also enhances BK Ca activity to induce hyperpolarization (43) and represses agonist-mediated Ca 2ϩ entry (30).…”