Imad S. Farrukh

Regulation of endothelin-1 synthesis in human pulmonary arterial smooth muscle cells effects of transforming growth factor-β and hypoxia

OBJECTIVE Endothelin-1 (ET-1) potently regulates pulmonary vascular tone and promotes vascular smooth muscle cell growth. Clinical and animal studies implicate increased ET-1 production in the pathogenesis of primary and secondary pulmonary hypertension. Although pulmonary arterial smooth muscle cells (PASMCs) synthesize ET-1 under basal conditions, it is unknown whether factors that may be important in pulmonary hypertension, such as transforming growth factor-beta (TGF-beta) or hypoxia, augment ET-1 production by these cells. METHODS We determined the effect of TGF-beta and hypoxia on ET-1 release and preproET-1 mRNA from cultured rat and human PASMCs. RESULTS In the basal state, rat and human PASMCs synthesize, on average (mean+/-S.E.M.), 872+/-114 and 563+/-57 pg ET-1/mg cell protein over 24 h, respectively, a level that causes autocrine and paracrine effects in other tissues. TGF-beta significantly increases the expression of preproET-1 mRNA and ET-1 production by both rat and human PASMCs. Hypoxia for 24 h, however, does not affect ET-1 release from rat or human PASMCs. CONCLUSIONS Cultured rat and human PASMCs are a source of ET-1 production. Enhanced ET-1 release from PASMCs may contribute to the pathophysiology of TGF-beta-induced pulmonary hypertension. ET-1 production by PASMCs is unlikely to contribute to the role of ET-1 in hypoxia-induced pulmonary vasoconstriction.

ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature

The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+ currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+ (KCa)-channel current [IK(Ca)] by increasing intracellular Ca2+ concentration, whereas it directly inhibits IK(Ca) in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced IK(Ca) inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces IK(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in IK(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in IK(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in IK(Ca) contributes to chronic hypoxia-induced pulmonary hypertension.The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+ currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+(KCa)-channel current [ I K(Ca)] by increasing intracellular Ca2+concentration, whereas it directly inhibits I K(Ca) in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced I K(Ca) inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces I K(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in I K(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in I K(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in I K(Ca)contributes to chronic hypoxia-induced pulmonary hypertension.

Role of Calcium ATPases in Pulmonary Vascular Reactivity

Changes in cytosolic Ca2+ [Ca2+]i primarily determine the contraction/relaxation cycle of vascular smooth muscle. Agonists that increase [Ca2+]i and induce vasoconstriction are counteracted by cellular Ca2+ transport mechanisms that decrease [Ca2+]i and modulate vasoconstriction. In vascular tissue, the Ca2+-ATPases play a crucial role in regulating [Ca2+]i and tone. Pulmonary vascular smooth muscle and endothelial cells possess two forms of Ca2+-ATPases: plasma membrane (PM) Ca2+ pumps (130–150 kd) and sarcoplasmic or endoplasmic reticulum (SER) Ca2+ pumps (100–115 kd) (12, 16, 17). The PM Ca2+ pumps along with Na+/Ca2+ exchange are the major mechanisms for Ca2+ extrusion from the cell. The SER Ca2+-ATPase pumps buffer an increase in [Ca2+]i by reloading intracellular stores with Ca2+ (16, 36).